GIFT  OF 


"Dr. 


BIOLOGY' 

LIBRARY 

G 


A  TEXT-BOOK 


UPON  THE 


PATHOGENIC  BACTERIA  - 


FOR 


STUDENTS  OF  MEDICINE  AND  PHYSICIANS 


BY 

JOSEPH  McFARLAND,  M.D. 

Demonstrator  of   Pathological    Histology   and    Lecturer   on   Bacteriology   in   the   Medical 
Department  of  the  University  of  Pennsylvania ;  Fellow  of  the  College  of 
Physicians  of  Philadelphia ;  Pathologist  to  the  Rush  Hos- 
pital for  Consumption  and  Allied  Diseases. 


WITH   113    ILLUSTRATIONS 


OF  THE 

UNIVERSITY 

OF 


PHILADELPHIA 

W.    B.    SAUNDERS 

925   WALNUT   STREET 
1896 


(V\3 

B/OLOGY 

LIBRARY 
Q 


Copyright,  1896,  by 
W.     B.    SAUNDERS. 


ELECTROTYPED    BY 


PRESS    OF 


WESTCOTT    &    THOMSON.   PHILADA.  w,   B.   SAUNDERS,    PHILAD/ 


Culture  of  glanders  bacillus  upon  cooked  potato  (Loffler). 


TO 
MY  HONORED  AND  BELOVED  GRANDFATHER 

MR.  JACOB   GRIM 

WHOSE   PARENTAL   LOVE  AND    LIBERALITY    HAVE   ENABLED   ME  TO  PURSUE 
MY    MEDICAL    EDUCATION 

4 

THIS    BOOK    IS    AFFECTIONATELY    DEDICATED 


PREFACE. 


THE  following  pages  are  intended  to  convey  to  the 
reader  a  concise  account  of  the  technical  procedures 
necessary  in  the  study  of  bacteriology,  a  brief  descrip- 
tion of  the  life-history  of  the  important  pathogenic 
bacteria,  and  sufficient  description  of  the  pathological 
lesions  accompanying  the  micro-organismal  invasions 
to  give  an  idea  of  the  origin  of  symptoms  and  the 
causes  of  death. 

The  work  being  upon  Pathogenic  Bacteria,  it  does 
not  cover  the  whole  scope  of  parasitology,  and  the 
parasites  of  higher  orders  are  all  omitted.  Malaria  and 
amebic  dysentery  are  omitted  as  logically  as  tape-worms 
and  pediculi.  The  higher  fungi  are  also  omitted,  both 
because  they  are  not  bacteria  and  because  their  proper 
consideration  would  make  a  small  book  in  itself. 

In  leaving  out  the  non-pathogenic  bacteria  of  course 
a  stumbling-block  was  encountered.  The  Sarciua  ven- 
triculi,  for  instance,  may  be  a  cause  of  dyspepsia,  yet 
can  scarcely  be  regarded  as  pathogenic,  and,  together 
with  other  similar  bacteria  of  questionable  deleterious 
operation,  has  been  omitted  ;  on  the  other  hand,  it 
has  been  thought  advisable  to  include  and  describe 
somewhat  at  length  a  long  list  of  spirilla  similar  to, 
and  probably  closely  allied  with,  the  spirillum  of 
cholera,  yet  not  the  cause  of  any  particular  diseased 

condition. 

11 


12  PREFACE. 

The  aim  has  been  to  describe  only  such  bacteria  as 
can  be  proven  pathogenic  by  the  lesions  or  toxins 
which  they  engender,  and,  while  considering  them,  to 
mention  as  fully  as  is  necessary  the  species  with  which 
they  may  be  confounded. 

The  book,  of  course,  will  find  its  proper  sphere  of 
usefulness  in  the  hands  of  medical  students  ;  its  pages, 
however,  will  be  found  to  contain  much  that  will  be 
of  interest  and  profit  to  those  practitioners  of  medicine 
who  graduated  before  modern  science  had  thrown  its 
light  upon  the  etiology  of  disease. 

In  writing  this  work  the  popular  text-books  have 
been  drawn  upon.  Hiippe,  Fliigge,  Sternberg,  Frankel, 
Giinther,  Thoinot  and  Masselin,  and  others  have  been 
freely  consulted. 

The  illustrations  are  mainly  reproductions  of  the  best 
the  world  affords,  and,  being  taken  from  the  great  stand- 
ards, are  surely  superior  to  anything  new  covering  the 
same  ground.  Credit  has  carefully  been  given  for  each 

illustration. 

J.  McF. 
PHILADELPHIA,  Feb.  i,  1896. 


CONTENTS. 


PART  I.    GENERAL  CONSIDERATIONS. 


PAGE 

INTRODUCTION 17 

CHAPTER  I. 
BACTERIA 30 

CHAPTER  II. 
THE  BIOLOGY  OF  BACTERIA 43 

CHAPTER  III. 
IMMUNITY  AND  SUSCEPTIBILITY 58 

CHAPTER  IV. 
METHODS  OF  OBSERVING  BACTERIA • 73 

CHAPTER  V. 
STERILIZATION  AND  DISINFECTION 90 

CHAPTER  VI. 
THE  CULTIVATION  OF  BACTERIA  ;  CULTURE-MEDIA 106 

CHAPTER  VII. 
CULTURES,  AND  THEIR  STUDY 117 

CHAPTER  VIII. 
THE  CULTIVATION  OF  ANAEROBIC  BACTERIA 130 

CHAPTER  IX. 
EXPERIMENTATION  UPON  ANIMALS 134 

13 


14  CONTENTS. 

CHAPTER  X. 

PAGE 

THE  RECOGNITION  OF  BACTERIA  ....      1.37 

CHAPTER  XL 
THE  BACTERIOLOGIC  EXAMINATION  OF  AIR 138 

CHAPTER  XII. 
THE  BACTERIOLOGIC  EXAMINATION  OF  WATER 143 

CHAPTER  XIII. 
THE  BACTERIOLOGIC  EXAMINATION  OF  SOIL 147 


PART  II.    SPECIFIC    DISEASES    AND    THEIR 
BACTERIA. 


A.  THE  PHLOGISTIC  DISEASES. 


I.  THE  ACUTE  INFLAMMATORY  DISEASES. 


CHAPTER  I. 
SUPPURATION 149 


II.  THE  CHRONIC  INFLAMMATORY  DISEASES. 


CHAPTER  I. 
TUBERCULOSIS  ....      169 

CHAPTER  II. 

IvEPROSY .    .  193 

CHAPTER  HI. 
GLANDERS 198- 

CHAPTER  IV. 
SYPHILIS 205 


CONTENTS.  15 

CHAPTER  V. 

PAGE 

ACTINOMYCOSIS 208 

CHAPTER  VI. 
MYCETOMA,  OR  MADURA-FOOT 212 

CHAPTER  VII. 
FARCIN  DU  BCEUF 216 

CHAPTER  VIII. 
RHINOSCLEROMA 219 


B.  THE  TOXIC  DISEASES. 


CHAPTER  I. 
DIPHTHERIA 220 

CHAPTER  II. 
TETANUS . • 235 

CHAPTER  III. 
HYDROPHOBIA,  OR  RABIES , 243 

CHAPTER  IV. 
SYMPTOMATIC  ANTHRAX 248 

CHAPTER  V. 
TYPHOID  FEVER 254 

CHAPTER  VI. 
CHOLERA 266 

CHAPTER  VII. 
SPIRILLA  RESEMBLING  THE  CHOLERA  SPIRILLUM 281 

CHAPTER  VIII. 
PNEUMONIA 297 


16  CONTENTS. 

C.  THE  SEPTIC  DISEASES. 


CHAPTER  I. 

PAGE 

RELAPSING  FEVER 307 

CHAPTER  II. 
INFLUENZA 309 

CHAPTER  III. 
MALIGNANT  EDEMA 312 

CHAPTER  IV. 
MEASLES 316 

CHAPTER  V. 
BUBONIC  PLAGUE 318 

CHAPTER  VI. 
TETRAGENUS 322 

CHAPTER  VII. 
CHICKEN-CHOLERA t 325 

CHAPTER  VIII. 
MOUSE-SEPTICEMIA 329 

CHAPTER  IX. 
ANTHRAX 334 

CHAPTER  X. 
TYPHUS  MURIUM 344 


INDEX 347 


PATHOGENIC    BACTERIA. 


PART   I.     GENERAL   CONSIDERATIONS. 


INTRODUCTION. 

THE  unrecognized  inception  of  the  department  of 
science  which  we  are  about  to  study  had  its  latent  germs 
in  the  thought  of  antiquity. 

It  is  folly  to  begin  the  consideration  of  bacteria  with 
their  probable  discoverer,  L,eeuwenhoek,  or  with  the  so- 
called  ' '  Father  of  bacteriology, ' '  Henle.  The  contro- 
versies and  ideas  which  stimulated  the  investigations  and 
researches  which  have  brought  us  to  our  present  state  of 
knowledge  were  begun  hundreds  of  years  before  the  be- 
ginning of  the  Christian  era. 

Excepting  such  as  taught  and  believed  that  * '  in  six 
days  the  Lord  made  heaven  and  earth,  the  sea  and  all 
that  in  them  is,"  or  a  kindred  theory  of  the  origin  of 
things,  the  thinkers  of  antiquity  never  seem  to  have 
doubted  that  under  favorable  conditions  life,  both  animal 
and  vegetable,  might  arise  spontaneously. 

Among  the  early  Greeks  we  find  that  Anaximander 
(43d  Olympiad,  610  B.  c.)  of  Miletus  held  the  theory  that 
animals  were  formed  from  moisture — an  idea  that  would 
stamp  him  a  disciple  of  Thales  if  we  did  not  know  that 
his  doctrine  was  that  u  the  Infinite  is  the  substance  of  all 
things."  Empedocles  of  Agrigentum  (4506.0.)  attrib- 
uted to  spontaneous  generation  all  the  living  beings 
which  he  found  peopling  the  earth.  Aristotle  (B.  c.  384) 
is  not  so  general  in  his  view  of  the  subject,  but  asserts 

2  17 


i8  :  ^PA  THOGENIC  BACTERIA. 

that  "sometimes  animals  are  formed  in  putrefying  soil, 
sometimes  in  plants,  and  sometimes  in  the  fluids  of  other 
animals."  He  also  formulated  a  principle  that  "every 
dry  substance  which  becomes  moist,  and  every  moist 
body  which  becomes  dried,  produces  living  creatures, 
provided  it  is  fit  to  nourish  them." 

Three  centuries  later,  in  his  disquisition  upon  the 
Pythagorean  philosophy,  we  find  Ovid  defending  the 
same  doctrine:1 

"By  this  sure  experiment  we  know 
That  living  creatures  from  corruption  grow  : 
Hide  in  a  hollow  pit  a  slaughter' d  steer, 
Bees  from  his  putrid  bowels  will  appear, 
Who,  like  their  parents,  haunt  the  fields  and  bring 
Their  honey-harvest  home,  and  hope  another  spring 
The  warlike  steed  is  multiplied,  we  find, 
To  wasps  and  hornets  of  the  warrior  kind. 
Cut  from  a  crab  his  crooked  claws,  and  hide 
The  rest  in  earth,  a  scorpion  thence  will  glide, 
And  shoot  his  sting  ;  his  tail  in  circles  toss'd 
Refers  the  limbs  his  backward  father  lost ; 
And  worms  that  stretch  on  leaves  their  filmy  loom 
Crawl  from  their  bags  and  butterflies  become. 
The  slime  begets  the  frog's  loquacious  race  ; 
Short  of  their  feet  at  first,  in  little  space, 
With  arms  and  legs  endued,  long  leaps  they  take, 
Raised  on  their  hinder  part,  and  swim  the  lake, 
And  waves  repel ;  for  nature  gives  their  kind, 
To  that  intent,  a  length  of  legs  behind." 

Not  only  was  the  doctrine  of  spontaneous  generation 
of  life  current  among  the  ancients,  but  we  find  it  persist- 
ing through  the  Middle  Ages,  and  descending  to  our  own 
generation  to  be  an  accidental  but  important  factor  in 
the  development  of  a  new  branch  of  science.  In  1542, 
in  his  treatise  called  De  Subtilitate,  we  find  Cardan  as- 
serting that  water  engenders  fishes,  and  that  many  ani- 
mals spring  from  fermentation.  Van  Helmont  gives 
special  instructions  for  the  artificial  production  of  mice, 

1  Ovid's  Metamorphoses,  translated  by  Mr.  Dryden,  published  by  Sir  Samuel 
Garth,  London,  1794. 


INTRODUCTION.  19 

and  Kirch er  in  his  Mundus  Subterraneus  (chapter  u  De 
Panspermia  Rerum  ")  describes  and  actually  figures  cer- 
tain animals  which  were  produced  under  his  own  eyes 
by  the  transforming  influence  of  water  on  fragments  of 
stems  from  different  plants.1 

About  1668,  Francesco  Redi  seems  to  have  been  the 
first  to  doubt  that  the  maggots  familiar  in  putrid  meat 
arose  de  novo :  "Watching  meat  in  its  passage  from 
freshness  to  decay,  prior  to  the  appearance  of  maggots, 
he  invariably  observed  flies  buzzing  around  the  meat  and 
frequently  alighting  on  it.  The  maggots,  he  thought, 
might  be  the  half-developed  progeny  of  these  flies. 
Placing  fresh  meat  in  a  jar  covered  with  paper,  he  found 
that  although  the  meat  putrefied  in  the  ordinary  way, 
it  never  bred  maggots,  while  meat  in  open  jars  soon 
swarmed  with  these  organisms.  For  the  paper  he  sub- 
stituted fine  wire  gauze,  through  which  the  odor  of  the 
meat  could  rise.  Over  it  the  flies  buzzed,  and  on  it  they 
laid  their  eggs,  but  the  meshes  being  too  small  to  per- 
mit the  eggs  to  fall  through,  no  maggots  generated  in 
the  meat;  they  were,  on  the  contrary,  hatched  on  the 
gauze.  By  a  series  of  such  experiments  Redi  destroyed 
the  belief  in  the  spontaneous  generation  of  maggots  in 
meat,  and  with  it  many  related  beliefs." 

It  was  not  long  before  Leeuwenhoek,  Vallismeri, 
Swammerdan,  and  others,  following  the  trend  of  Redi's 
work,  contributed  additional  facts  in  favor  of  his  view, 
and  it  may  safely  be  asserted  that  ever  since  the  time 
of  this  eminent  man  the  tide  of  scientific  opinion  has 
turned  more  and  more  strongly  against  the  idea  that 
life  is  spontaneously  generated. 

About  this  time  (1675)  one  whose  name  has  been 
already  mentioned,  Anthony  van  Leeuwenhoek,  and  who 
is  justly  called  the  "Father  of  microscopy,"  came  into 
prominence.  An  optician  by  trade,  Leeuwenhoek  devoted 
much  time  to  the  perfection  of  the  compound  micro- 
scope, which  was  just  coming  into  use.  The  science  of 

1  See  Tyndall :  Floating  Matter  in  the  Air. 


20  PA  THOGENIC  BA  CTERIA . 

optics,  however,  was  not  sufficiently  developed  to  enable 
him  to  overcome  the  errors  of  refraction,  and  after  the 
loss  of  much  time  he  turned  to  the  simple  lens,  using  it 
in  so  careful  and  remarkable  a  manner  as  to  be  able 
to  record  his  observations  in  one  hundred  and  twelve 
contributions  to  the  Philosophical  Transactions.  L,eeu- 
wenhoek,  among  other  things,  demonstrated  the  conti- 
nuity of  arteries  and  veins  through  intervening  capil- 
laries, thus  affording  ocular  proof  of  Harvey's  discovery 
of  the  circulation  of  the  blood;  discovered  the  rotifers, 
and  also  the  bacteria,  seeing  them  first  in  saliva. 

Although  one  of  those  who  contributed  to  the  support 
of  Redi's  arguments  against  the  spontaneous  generation 
of  maggots,  lyeeuwenhoek  involuntarily  reopened  the  old 
controversy  about  spontaneous  generation  by  bringing 
forward  a  new  world,  peopled  by  creatures  of  such  ex- 
treme minuteness  as  to  suggest  not  only  a  close  relation- 
ship to  the  ultimate  molecules  of  matter,  but  an  easy 
transition  from  them. 

In  succeeding  years  the  development  of  the  compound 
microscope  showed  these  minute  organisms  to  exist  in 
such  numbers  that  putrescent  infusions,  both  animal  and 
vegetable,  literally  teemed  with  them,  one  drop  of  such 
a  liquid  furnishing  a  banquet  for  millions. 

Much  hostility  arose  in  the  scientific  world  as  years 
went  on  until  two  schools  attained  prominence — one 
headed  by  Buffon,  whose  doctrine  was  that  of  "organic 
molecules;"  the  other  championed  by  Needham,  whose 
doctrine  was  the  existence  of  a  "vegetative  force"  which 
drew  the  molecules  together. 

Experimentation  was  begun  and  attracted  much  atten- 
tion. Among  the  pioneers  was  Abbe  Lazzaro  Spallan- 
zani  (1777),  who  filled  flasks  with  organic  infusions, 
sealed  their  necks,  and,  after  subjecting  their  contents 
to  the  temperature  of  boiling  water,  placed  them  under 
conditions  favorable  for  the  development  of  life,  without, 
however,  being  able  to  produce  it.  Spallanzani's  critics, 
however,  objected  to  his  experiment  on  the  ground  that 


INTRODUCTION.  21 

air  is  essential  to  life,  and  that  in  his  flasks  the  air  was 
excluded  by  the  hermetically-sealed  necks. 

Schulze  (1836)  set  the  objection  aside  by  filling  a  flask 
only  half  full  of  distilled  water,  to  which  animal  and 
vegetable  matters  were  added,  boiling  the  contents  to 
destroy  the  vitality  of  any  organisms  which  might  al- 
ready exist  in  them,  then  sticking  daily  into  the  flask  a 
certain  amount  of  air  which  had  passed  through  a  series 
of  bulbs  containing  concentrated  sulphuric  acid,  in  which 
it  was  supposed  that  whatever  germs  of  life  the  air  might 
contain  would  be  destroyed.  This  flask  was  kept  from 
May  to  August;  air  was  passed  through  it  daily,  yet  with- 
out the  development  of  any  infusorial  life.- 

The  term  "infusorial  life"  having  been  used,  here  it 
is  well  to  observe  that  during  all  the  early  part  of  their 
recognized  existence  the  bacteria  were  regarded  as  ani- 
mal organisms  and  classed  among  the  infusoria. 

Cagniard  Latour  and  Schwann  in  the  year  1837  suc- 
ceeded in  proving  that  the  minute  oval  bodies  which  had 
been  observed  in  yeast  since  the  the  time  of  Leeuwenhoek 
were  living  organisms — vegetable  forms — capable  of 
growth ;  and  when  Boehm  succeeded  a  year  later  in  de- 
monstrating their  occurrence  in  the  stools  of  cholera,  and 
conjectured  that  the  process  of  fermentation  was  con- 
cerned in  the  causation  of  that  disease,  the  study  of  these 
low  forms  of  life  received  an  immense  impetus  from  the 
important  position  which  they  began  to  assume  in  rela- 
tion to  medical  science. 

The  experiments  of  Schwann,  by  proving  that  the 
free  admission  of  calcined  air  to  closed  vessels  contain- 
ing putrescible  infusions  was  without  effect,  while  the 
admission  of  ordinary  air  brought  about  decomposition, 
suggested  that  the  causes  of  putrefaction  which  were  in 
the  air  were  living  entities. 

In  1862,  Pasteur  published  a  paper  "  On  the  Organized 
Corpuscles  existing  in  the  Atmosphere,"  in  which  he 
showed  that  many  of  the  floating  particles  which  he 
had  been  able  to  collect  from  the  atmosphere  of  his 


22  PA  THOGENIC  BA CTERIA. 

laboratory  were  organized  bodies.  If  these  were  planted 
in  sterile  infusions,  abundant  crops  of  micro-organisms 
were  obtainable.  By  the  use  of  more  refined  methods 
he  repeated  the  experiments  of  Schwann  and  others,  and 
showed  clearly  that  "the  cause  which  communicated  life 
to  his  infusions  came  from  the  air,  but  was  not  evenly  dis- 
tributed through  it." 

Three  years  later  he  showed  that  the  organized  cor- 
puscles which  he  had  found  in  the  air  were  the  spores  or 
seeds  of  minute  plants,  and  that  many  of  them  possessed 
the  property  of  withstanding  the  temperature  of  boiling 
water — a  property  which  explained  the  peculiar  results 
of  many  previous  experimenters,  who  failed  to  prevent 
the  development  of  life  in  boiled  liquids  enclosed  in 
hermetically-sealed  flasks. 

Chevreul  and  Pasteur  (about  1836)  proved  that  animal 
solids  did  not  putrefy  or  decompose  if  kept  free  from 
the  access  of  germs,  and  thus  suggested  to  surgeons  that 
the  putrefaction  which  occurred  in  wounds  was  due  rather 
to  the  entrance  of  something  from  without  than  to  some 
change  within.  The  deadly  nature  of  the  discharges 
from  these  wounds  had  been  shown  in  a  rough  manner 
by  Gaspard  as  early  as  1822  by  injecting  some  of  the 
material  into  the  veins  of  animals. 

Examinations  of  the  blood  of  diseased  animals  were 
now  begun,  and  Pollender  (1849)  and  Davaine  (1850) 
succeeded  in  demonstrating  the  presence  of  the  anthrax 
bacillus  in  that  disease.  Several  years  later  (1863)  Da- 
vaine,  having  made  numerous  inoculation-experiments, 
demonstrated  that  this  bacillus  was  the  materies  morbi 
of  the  disease. 

Tyndall  enlarged  upon  the  experiments  of  Pasteur, 
and  very  conclusively  proved  that  the  micro-organ ismal 
germs  were  in  the  dust  suspended  in  the  atmosphere,  not 
ubiquitous  in  their  distribution.  His  experiments  were 
very  ingenious  and  are  of  interest  to  medical  men.  First 
preparing  light  wooden  chambers,  with  one  large  glass 
window  in  the  front  and  one  smaller  window  in  each 


INTRODUCTION.  23 

side,  he  arranged  a  series  of  empty  test-tubes  in  the 
bottom  and  a  pipette  in  the  top,  so  that  when  desired 
the  tubes,  one  by  one,  could  be  filled  through  it.  The 
chamber  was  first  submitted  to  an  optical  test  to  deter- 
mine the  purity  of  its  atmosphere,  and  was  allowed  to 
stand  undisturbed  and  unused  until  a  powerful  ray  of 
light  passed  through  the  side  windows  failed  to  reflect 
rays  from  suspended  particles  of  dust  when  viewed  from 
the  front.  When  the  dust  had  settled  so  as  to  allow  the 
optical  test  of  its  purity,  the  tubes  were  filled  with  urine, 
beef-broth,  and  a  variety  of  animal  and  vegetable  broths, 
boiled  by  submergence  in  a  pan  of  hot  brine;  the  tubes 
were  then  allowed  to  remain  undisturbed  for  days,  weeks, 
or  months.  In  nearly  every  case  life  failed  to  develop 
after  the  purity  of  the  atmosphere  was  established. 

In  1873,  Obermeier  observed  that  actively  motile,  flex- 
ible spiral  organisms  were  present  in  large  numbers  in 
the  blood  of  patients  in  the  febrile  stages  of  relapsing 
fever. 

Thus  evidence  slowly  accumulated  to  establish  the 
theory  for  which  Henle  had  labored  as  early  as  1821,  that 
for  many  diseases  at  least  there  was  a  distinct  and  specific 
contagium  vivzim,  and  the  ' '  GERM  THEORY ' '  was  pro- 
pounded. 

Is  it  not  strange  that  the  very  idea  which  was  to  be  the 
outcome  of  all  this  investigation  and  discussion — an  idea 
which  would  form  a  new  era  in  scientific  medicine  and 
become  a  fundamental  principle  of  pathology — was  one 
which  had  been  conceived  and  taught  by  a  philosopher 
who  lived  nearly  two  thousand  years  ago  ?  Among  the 
numerous  works  of  Varro *  is  one  entitled  Rerum  Rusti- 
carum  libri  tres,  from  which  the  following  is  quoted  : 
uAnimadvertendum  etiam,  si  qua  erunt  loca  palustria — 
quod  crescunt  animalia  quaedam  minuta,  quae  non  pos- 
sunt  oculi  consequi  et  per  ae'ra  intus  in  corpus  per  os  ac 
nares  perveniunt  atque  eificiunt  difficilis  morbus "  (I., 
xii.  2). — u  It  is  also  to  be  noticed,  if  there  be  any  marshy 

1  Univ.  Med.  Mag.,  vol.  iii.,  No.  3,  Dec.,  1890,  p.  152. 


24  PATHOGENIC  BACTERIA. 

places,  that  certain  minute  animals  breed  [there]  which 
are  invisible  to  the  eye,  and  yet,  getting  into  the  sys- 
tem through  mouth  and  nostrils,  cause  serious  disor- 
ders (diseases  which  are  difficult  to  treat)" — a  doctrine 
which,  as  Prof.  L,amberton,  to  whom  the  writer  is  in- 
debted for  the  extract,  points  out,  is  handed  down  to  us 
from  "the  days  of  Cicero  and  Caesar,"  yet  corresponds 
closely  to  the  ideas  of  malaria  which  we  entertain  at 
present. 

Pasteur  had  long  before  suggested  that  for  the  different 
kinds  of  fermentation  there  must  be  specific  ferments, 
and  by  fractional  cultures  had  succeeded  in  roughly  sepa- 
rating them. 

Klebs,  who  was  one  of  the  pioneers  of  the  germ 
theory,  published  in  1872  his  work  upon  septicemia  and 
pyemia,  in  which  he  expressed  himself  convinced  that 
the  causes  of  these  diseases  must  come  from  without  the 
body.  Billroth  strongly  opposed  such  an  idea,  asserting 
that  fungi  had  no  especial  importance  either  in  the  pro- 
cesses of  disease  or  in  those  of  decomposition,  but  that, 
existing  everywhere  in  the  air,  they  rapidly  developed  in 
the  body  as  soon  as  through  putrefaction  a  ' '  Faulniss- 
zymoid,"  or  through  inflammation  a  "  phlogistische- 
zymoid,"  supplying  the  necessary  feeding-grounds,  was 
produced. 

Klebs  was  not  alone  in  the  opposition  aroused.  Da- 
vaine  no  sooner  announced  the  contagium  of  anthrax 
than  critics  declared  that  inasmuch  as  he  introduced 
blood  from 'the  diseased  animal  into  the  other  animal 
to  whom  the  disease  was  to  be  communicated,  it  was 
altogether  unreasonable  to  believe  the  bacilli  which  were 
in  all  probability  accidentally  present  in  that  blood  were 
the  cause  of  the  disease. 

In  1875  the  number  of  scientific  men  who  had  embraced 
the  germ  theory  of  disease  was  small,  and  most  of  those 
who  accepted  it  were  experimenters.  A  great  majority 
of  medical  men  either  believed,  like  Billroth,  that  the 
presence  of  fungi  where  decomposition  was  in  progress 


INTRODUCTION.  25 

was  an  accidental  result  of  their  universal  distribution, 
or,  being  still  more  conservative,  retained  the  old  un- 
questioning faith  that  the  bacteria,  whose  presence  in 
putrescent  wounds  as  well  as  in  artificially  prepared 
media  was  unquestionable,  were  spontaneously  generated 
there. 

The  following  extracts  from  Tyndall's  work1  will  illus- 
trate the  slow  growth  of  the  germ  theory  even  among 
men  of  eminence  : 

u  At  a  meeting  of  the  Pathological  Society  of  London, 
held  April  6,  1875,  the  *  germ  theory '  of  disease  was 
formally  introduced  as  a  subject  for  discussion,  the  debate 
being  continued  with  great  ability  and  earnestness  at  sub- 
sequent meetings.  The  conference  was  attended  by 
many  distinguished  medical  men,  some  of  whom  were 
profoundly  influenced  by  the  arguments,  and  none  of 
whom  disputed  the  facts  brought  forward  against  the 
theory  on  that  occasion." 

"The  leader  of  the  debate,  and  the  most  prominent 
speaker,  was  Dr.  Bastian,  to  whom  also  fell  the  task  of 
replying  on  all  the  questions  raised." 

"The  coexistence  of  bacteria  and  contagious  disease 
was  admitted ;  but,  instead  of  considering  these  organisms 
as  probably  the  essence,  or  an  inseparable  part  of  the  es- 
sence, of  the  contagium,  Dr.  Bastian  contended  that  they 
were  pathological prodticts  spontaneously  generated  in  the 
body  after  it  had  been  rendered  diseased  by  the  real  con- 
tagium. ' ' 

"The  grouping  of  the  ultimate  particles  of  matter  to 
form  living  organisms  Dr.  Bastian  considered  to  be  an 
operation  as  little  requiring  the  action  of  antecedent  life 
as  their  grouping  to  form  any  of  the  less  complex  chem- 
ical compounds. "  "  Such  a  position  must,  of  course, 
stand  or  fall  by  the  evidence  which  its  supporter  is  able 
to  produce,  and  accordingly  Dr.  Bastian  appeals  to  the 
law  and  testimony  of  experiment  as  demonstrating  the 
soundness  of  his  view."  "  He  seems  quite  aware  of  the 

1  Op.  cit. 


26  PATHOGENIC  BACTERIA. 

gravity  of  the  matter  at  hand  ;  this  is  his  deliberate  and 
almost  solemn  appeal  :  '  With  the  view  of  settling  these 
questions,  therefore,  we  may  carefully  prepare  an  infusion 
from  some  animal  tissue,  be  it  muscle,  kidney,  or  liver  ; 
we  may  place  it  in  a  flask  whose  neck  is  drawn  out 
and  narrowed  in  the  blowpipe  flame ;  we  may  boil  the 
fluid,  seal  the  vessel  during  ebullition,  and,  keeping  it 
in  a  warm  place,  may  await  the  result,  as  I  have  often 

done After  a  variable  time  the  previously  heated 

fluid  within  the  hermetically-sealed  flask  swarms  more 
or  less  plentifully  with  bacteria  and  allied  organisms, 
even  though  the  fluids  have  been  so  much  degraded  in 
quality  by  exposure  to  the  temperature  of  212°  F.,  and 
have  in  all  probability  been  rendered  far  less  prone  to 
engender  independent  living  units  than  the  unheated 
fluids  in  the  tissues  would  be.'  ' 

These  somewhat  lengthy  quotations  are  of  great  in- 
terest, for  they  show  exactly  the  state  of  the  scientific 
mind  at  a  period  as  recent  as  twenty  years  ago. 

In  1877  the  introduction  of  the  anilin  dyes  by  Weigert 
made  possible  a  much  more  thorough  investigation  of 
the  bacteria  by  enabling  the  observers  to  color  them 
intensely,  and  thus  detect  their  presence  in  tissues  and 
organs  where  their  transparency  had  caused  them  to  be 
overlooked. 

Rapid  strides  were  immediately  made,  and  before 
another  decade  had  passed  discoveries  were  so  numerous 
and  convincing  that  it  was  impossible  to  doubt  that  bac- 
teria were  causes  of  disease. 

Before  the  publication  of  the  discoveries  of  which  we 
speak,  however,  there  was  suggested  a  practical  applica- 
tion of  the  little  known  about  bacteria  which  produced 
greater  agitation  and  incited  more  observation  and  ex- 
perimentation than  anything  suggested  in  surgery  since 
the  introduction  of  anesthetics — namely,  antisepsis. 

u  The  seminal  thought  of  antiseptic  surgery  may  per- 
haps be  traced  to  John  Colbach,  a  member  of  the  College 
of  Physicians,  England,  whose  collection  of  tracts,  printed 


INTRODUCTION.  27 

1 704,  contained  a  description  of  a  new  and  secret  method 
of  treating  wounds,  by  which  healing  took  place  quickly 
without  inflammation  or  suppuration ;  but  it  is  to  one  of 
old  Scotia's  sons,  Sir  Joseph  lyister,  that  the  everlasting 
gratitude  of  the  world  is  due  for  the  knowledge  we  pos- 
sess in  regard  to  the  relation  existing  between  micro- 
organisms and  inflammation  and  suppuration,  and  the 
power  to  render  wounds  aseptic  through  the  action  of 
germicidal  substances."  l 

Lister  was  not  the  discoverer  of  carbolic  acid  nor  of 
the  fact  that  it  would  kill  bacteria;  but,  convinced  that 
inflammation  and  suppuration  were  due  to  the  entrance 
of  germs  from  the  air,  instruments,  fingers,  etc.  into 
wounds,  he  suggested  the  antisepsis  which  would  insist 
upon  the  use  of  sterile  instruments  and  clean  hands  and 
towels;  which  would  keep  the  surface  of  the  wound 
moist  with  a  germicidal  solution  to  kill  such  germs  as 
accidentally  entered;  and  which  would  conclude  an  ope- 
ration by  a  protective  dressing  to  exclude  the  entrance  of 
germs  at  a  subsequent  period. 

Listerism,  originated  (1875)  a  few  years  before  Koch 
published  his  famous  work  on  the  Wundinfectionskrank- 
heiten  (traumatic  infectious  diseases)  (1878),  spread  slowly 
at  first,  but  surely  in  the  end,  to  all  departments  of  sur- 
gery and  obstetrics. 

The  discovery  of  the  yeast-plant  by  Latour  and 
Schwann  as  the  cause  of  fermentation,  and  the  later  dis- 
covery by  Bassi  of  the  yeast-like  plant  causing  the  mias- 
matic contagious  disease  of  silkworms,  had  led  Henle 
(1840)  to  believe  that  the  cause  of  miasmatic,  infective, 
and  contagious  diseases  must  be  looked  for  in  fungi  or 
in  other  minute  living  organisms.  Unfortunately,  the 
methods  of  study  employed  in  Henle' s  time  prevented 
him  from  demonstrating  the  accuracy  of  his  belief. 

"It  would  indeed  have  been  difficult  at  that  period  to 
satisfy  every  condition  that  he  required  to  be  fulfilled: 
the  methods  now  in  use  were  then  unknown,  and  have 

1  Agnew's  Surgery,  vol.  i.  chap.  ii. 


28  PATHOGENIC  BACTERIA. 

only  been  perfected  by  workers  as  it  has  been  found  nec- 
essary from  time  to  time  to  comply  in  the  most  minute 
detail  with  Henle's  conditions,  and  as,  one  point  being 
carried,  it  was  found  necessary  to  advance  on  others. 
The  first  of  these  was  that  a  specific  organism  should 
always  be  associated  with  the  disease  under  consideration. 
As  such  presence,  however,  might  be  accidental,  these 
organisms  were  not  only  to  be  found  in  pus,  etc. ,  but  actu- 
ally in  the  living  body.  As  they  might  be,  even  then, 
merely  parasitic,  and  not  associated  directly  with  the 
causation  of  the  disease,  it  would  be  necessary  to  isolate 
the  germs,  the  contagium  organisms,  and  the  contagium 
fluids,  and  to  experiment  with  these  separately  with 
special  reference  to  their  power  of  producing  similar 
diseases  in  other  animals.  We  now  know  that  it  has 
only  been  by  strict  compliance  with  all  these  conditions, 
again  postulated  by  Koch,  that  the  most  brilliant  scien- 
tific observers  and  experimentalists  in  Germany,  France, 
England,  [and  America]  have  been  able  to  determine 
the  causal  connection  between  micro-organisms  and 
disease."1 

The  refined  methods  of  Pasteur,  but  more  especially 
of  Koch,  by  making  possible  the  fulfilment  of  the  pos- 
tulates of  Henle  caused  an  enormous  increase  in  the 
rapidity  with  which  data  upon  disease-germs  were  gath- 
ered. Almost  within  a  decade  the  causes  of  the  most 
important  specific  diseases  were  isolated  and  cultivated. 

In  1879,  Hausen  announced  the  discovery  of  bacilli  in 
the  cells  of  leprous  nodules.  The  same  year  Neisser 
discovered  the  gonococcus  to  be  specific  for  gonorrhea. 

In  1880  the  bacillus  of  typhoid  fever  was  first  observed 
by  Eberth,  and  independently  by  Koch. 

In  1880,  Pasteur  published  his  work  upon  "chicken- 
cholera."  In  the  same  year  Sternberg  described  the 
pneumococcus,  calling  it  the  micrococcus  Pasteuri. 

In  1882,  Koch  made  himself  immortal  by  his  discov- 
ery of  and  work  upon  the  tubercle  bacillus.  The  same 

1  Woodhead  :   Bacteria  and  their  Products,  p.  65. 


INTRODUCTION.  29 

year  Pasteur  published  a  work  upon  Rouget  du  Pore,  and 
Iv6ffler  and  Schiitz  reported  the  discovery  of  the  bacillus 
of  glanders. 

In  1884,  Koch  reported  the  discovery  of  the  "comma 
bacillus,"  the  cause  of  cholera,  and  in  the  same  year 
Iv6ffler  discovered  the  diphtheria  bacillus,  and  Nicolaier 
the  tetanus  bacillus. 

In  1892,  Canon  and  Pfeiffer  discovered  the  bacillus  of 
influenza. 

In  1892,  Canon  and  Pielicke  first  found  the  bacillus 
now  thought  to  be  specific  for  measles. 

In  1894,  Yersin  and  Kitasato  independently  isolated 
the  bacillus  causing  the  bubonic  plague  then  prevalent 
at  Hong-Kong. 

Between  the  years  1884  and  1892  few  new  bacteria 
were  discovered,  attention  being  directed  toward  perfect- 
ing the  methods  of  technical  procedure,  investigating 
interesting  subjects  relating  to  the  biology  of  the  bac- 
teria, and  the  study  of  immunity. 


CHAPTER  I. 
BACTERIA. 

A  BACTERIUM  is  a  minute  vegetable  organism  consist- 
ing of  a  single  cell  principally  composed  of  an  albumin- 
ous substance,  which  Nencki  has  called  mycoprotein. 
Nencki  found  the  chemical  analysis  of  bacteria  in  the 
active  state  to  consist  of  82.42  per  cent,  of  water.  In 
i oo  parts  of  the  dried  constituents  he  found  84.20  parts 
of  mycoprotein;  6.04  of  fat;  4.72  of  ash;  5.04  of  unde- 
termined substances. 

Mycoprotein,  which  has  the  composition  C  52.32,  H 
7.55,  N  14.75,  is  a  perfectly  transparent,  generally  ho- 
mogeneous body,  which  probably  varies  somewhat  ac- 
cording to  the  species  from  which  it  is  obtained,  the 
culture-medium  in  which  it  is  grown,  and  the  vital 
products  which  the  organism  produces  by  its  growth. 
Sometimes  the  mycoprotein  is  granular,  as  in  bacillus 
megatherium  ;  sometimes  it  contains  fine  granules  of 
chlorophyl,  sulphur,  fat,  or  pigment.  Each  cell  is  sur- 
rounded by  a  cell-wall,  which  in  some  species  shows  the 
cellulose  reaction  with  iodin. 

When  subjected  to  the  influence  of  nuclear  stains  the 
bacteria  not  only  take  the  stain  faintly,  but  in  such  a 
manner  as  to  show  the  existence  of  a  large  nucleus  situ- 
ated in  the  centre  of  the  cell  and  constituting  its  great 
bulk.  The  cell-wall  generally  is  not  stained,  but  when 
it  does  tinge,  a  delicate  line  of  unstained  material  can 
sometimes  be  made  out  between  the  nucleus  and  the  cell- 
wall,  showing  the  existence  of  a  protoplasm. 

The  anilin  dyes,  which  possess  a  great  penetrating 
power,  color  the  organisms  so  intensely  as  to  preclude 
the  differentiation  of  the  cellular  constituents.  Under 

30 


BACTERIA.  31 

these  conditions  the  bacteria  appear  as  solidly-colored 
spheres,  rods,  or  spirals,  as  the  case  may  be. 

The  cell-walls  of  some  of  the  bacteria  seem  at  times  to 
undergo  a  peculiar  gelatinous  change  or  to  allow  the  ex- 
udation of  gelatinous  material  from  the  protoplasm,  so 
that  the  individuals  appear  surrounded  by  a  distinct  halo. 
This  is  not  only  a  peculiarity  of  certain  individuals,  but 
one  which  only  takes  place  when  they  develop  under 
certain  conditions;  thus,  Friedlander  points  out  that  the 
capsule  of  his  pneumonia  bacillus,  when  it  was  found  in 
the  lung  or  in  the  "prune-juice"  sputum,  was  very  dis- 
tinct, while  it  could  not  be  demonstrated  at  all  when  the 
organisms  grew  in  gelatin. 

From  the  cell-walls  of  many  bacteria  numerous  deli- 
cate straight  or  wavy  filaments  project.  These  are  called 
cilia  or  flagella,  and  seem  to  be  organs  of  locomotion. 
Sometimes  they  are  only  observed  projecting  from  the 
ends  or  from  one  end;  sometimes  they  are  so  numerous 
and  so  regular  in  their  distribution  as  to  give  the  organ- 
isms a  woolly  appearance. 

Many  of  the  bacteria  which  are  thus  supplied  with 
flagella  are  actively  motile  and  swim  about  like  mi- 
croscopic serpents.  In  all  probability  the  locomotory 
powers  of  the  bacteria  are  not  entirely  dependent  upon 
the  presence  of  the  flagella,  but  may  sometimes  be  due 
to  contractility  of  the  protoplasm  within  an  elastic  cell- 
wall.  The  micro-organisms  most  plentifully  supplied 
with  them  are  those  of  the  rod  and  spiral  shape.  Only 
one  of  the  spherical  forms,  Micrococcus  agilis  of  Ali- 
Cohen,  has  been  shown  to  have  flagella.  This  and  one 
other  species  are  probably  the  only  motile  cocci.  Ob- 
serving that  the  organisms  known  to  be  most  active  are 
those  best  supplied  with  flagella,  it  is  reasonable  to  con- 
clude that  the  motility  is  dependent  upon  the  flagella. 

The  presence  of  flagella,  however,  does  not  necessarily 
imply  motility,  for  some  of  the  bacilli  amply  provided 
with  these  appendages  are  not  motile  (bacillus  coli  com- 
munis).  The  flagella  may  not  only  serve  as  organs  of 


32  PATHOGENIC  BACTERIA. 

locomotion,  and  be  of  use  to  the  organism  by  conveying 
it  from  an  area  where  the  nutrition  is  less  to  one  where 
it  is  greater,  but,  as  Woodhead  points  out,  may,  in  the 
non-motile  species,  serve  to  stimulate  the  passage  of  cur- 
rents of  nutrient  material  past  the  organism,  so  as  to  in- 
crease the  food-supply.  The  flagellate  bacteria  have  a 
greater  number  of  representatives  among  those  whose 
lives  are  spent  in  water  and  in  fermenting  and  decaying 
materials  than  among  those  inhabiting  the  bodies  of 
animals.  This  is  an  additional  fact  in  favor  of  the  view 
that  locomotion  and  flagella  are  provisions  favorable  to 
the  maintenance  of  the  species  by  keeping  the  individuals 
supplied  with  food. 

In  carrying  the  argument  a  little  farther  it  may  be 
added  that  such  parasitic  disease-producing  bacteria  as  do 
not  habitually  gain  access  to  the  tissues,  but  inhabit  the 
intestine,  as  the  bacillus  of  typhoid  fever  and  the  spirillum 
of  cholera,  are  actively  motile,  like  the  saprophytes.  Of 
course  this  example  is  open  to  criticism,  because  the  spi- 
rillum of  relapsing  fever,  which  has  never  been  found 
elsewhere  than  in  the  blood  and  spleen  of  affected  ani- 
mals, is  actively  motile,  while  the  Bacterium  coli  com- 
munis,  which  is  always  present  in  the  intestine,  is  non- 
motile. 

One  of  the  linear  organisms  known  as  the  Bacillus  meg- 
atherium has  a  distinct  but  limited  ameboid  movement. 

The  commonly  observed  dancing  movement  of  the 
spherical  forms  seems  to  be  the  well-known  Brownian 
movement,  which  is  simply  a  physical  phenomenon.  It 
is  sometimes  difficult  to  determine  whether  an  organism 
is  really  motile  or  whether  it  is  only  vibrating.  In  the 
latter  case  it  does  not  change  its  relative  position  to 
surrounding  objects. 

The  bacteria  are  so  minute  that  a  special  unit  of  meas- 
urement has  been  adopted  by  bacteriologists  for  their 
estimation.  This  is  the  micro-millimeter  (//),  or  one- 
thousandth  part  of  a  millimeter,  and  about  equivalent 
to  the  one-twenty-five-thousandth  of  an  inch. 


BACTERIA.  33 

As  a  rule,  the  spherical  organisms  are  the  smallest  and 
the  spiral  organisms  the  longest,  except  the  chains  of 
bacilli  called  leptothrix.  Their  measurements  vary  from 
o.  15  f*  (micrococcus  of  progressive  abscess- formation  in 
rabbits)  to  2.8  ft  (Diplococcus  albicans  am  plus)  for  cocci, 
and  from  i  X  0.2  //  (bacillus  of  mouse-septicemia)  to 
5  X  1.5  ft  (anthrax  bacillus)  for  bacilli.  Some  of  the 
spirilla  are  very  long,  that  of  relapsing  fever  measuring 
40  fJ.  at  times. 

This  estimation  of  size  almost  prepares  one  for  the 
estimation  of  weight  given  by  Nageli,  who  found  that 
an  average  bacterium  under  ordinary  conditions  weighed 
iOoooooTQOT  of  a  milligram. 

The  bacteria  multiply  in  two  ways  :  by  direct  division 
(fission)  and  by  the  development  of  spores,  seeds,  or  eggs 
(sporulation).  The  more  common  mode  is  by  binary 
division.  The  bacterium  which  is  about  to  divide  ap- 
pears a  little  larger  than  normal,  and,  if  a  spherical 
organism,  more  or  less  ovoid.  No  karyokinetic  changes 
have  been  observed  in  the  nuclei,  though  they  may  occur. 
When  the  conditions  of  nutrition  are  good,  the  process  of 
fission  progresses  with  astonishing  rapidity.  Buchner 
and  others  have  determined  the  length  of  a  generation 
to  be  from  fifteen  to  forty  minutes. 

The  results  of  binary  division,  if  rapidly  repeated,  are 
almost  appalling.  "  Cohn  calculated  that  a  single  germ 
could  produce  by  simple  fission  two  of  its  kind  in  an 
hour  ;  in  the  second  hour  these  would  be  multiplied  to 
four  ;  and  in  three  days  they  would,  if  their  surroundings 
were  ideally  favorable,  form  a  mass  which  can  scarcely  be 
reckoned  in  numbers,  or,  if  reckoned,  could  scarcely  be 
imagined — four  thousand  seven  hundred  and  seventy-two 
billions.  If  we  reduce  this  number  to  weight,  we  find 
that  the  mass  arising  from  this  single  germ  would  in 
three  days  weigh  no  less  than  seventy-five  hundred 
tons."  "Fortunately  for  us,"  says  Woodhead,  uthey 
can  seldom  get  food  enough  to  carry  on  this  appalling 
rate  of  development,  and  a  great  number  die  both  for 


34  PATHOGENIC  BACTERIA. 

want  of  food  and  because  of  the  presence  of  other  con- 
ditions unfavorable  to  their  existence." 

When  the  conditions  for  rapid  multiplication  are  no 
longer  good,  the  organism  assumes  a  protective  attitude 
and  develops  in  its  interior  small  oval  eggs,  seeds,  or,  as 
they  are  more  correctly  called,  spores  (Fig.  i).  Such 

a  b  c  d  e  f 

.CJED          c:  —  g)          o         o  o 


FIG.  I.  —  Diagram  illustrating  sporulation  :  a,  bacillus  enclosing  a  small  oval 
spore  ;  b,  drumstick  bacillus,  with  the  spore  at  the  end  ;  c,  clostridium  ;  d,  free 
spores  ;  e  and  fy  bacilli  escaping  from  spores. 

spores  developed  within  the  bacteria  are  called  endospores. 
When  the  formation  of  such  a  spore  is  about  to  com- 
mence, a  small  bright  point  appears  in  the  protoplasm, 
and  increases  in  size  until  its  diameter  is  nearly  or  quite 
as  great  as  that  of  the  bacterium.  As  it  nears  perfection 
a  dark,  highly-refracting  capsule  is  formed  about  it.  As 
soon  as  the  spore  arrives  at  perfection  the  bacterium 
seems  to  die,  as  if  its  vitality  were  exhausted  in  the 
development  of  the  permanent  form. 

Endospores  are  generally  formed  in  the  elongate  bac- 
teria —  bacillus  and  spirillum  —  but  Zopf  has  described 
similar  bodies  as  occurring  in  micrococci.  Escherich 
also  claims  to  have  found  undoubted  spores  in  a  form 
of  sarcina. 

The  spores  found  in  the  bacilli  are  either  round  or 
oval.  As  a  rule,  each  bacillus  produces  a  single  spore, 
which  is  situated  either  at  its  centre  or  at  its  end.  When, 
as  sometimes  happens,  the  diameter  of  the  spore  is  greater 
than  the  diameter  of  the  bacillus,  it  causes  a  bulging  of 
the  organism,  with  a  peculiar  appearance  described  as 
clostridium.  When  the  distending  spore  is  in  the  centre 
of  the  bacillus,  it  produces  a  barrel-shaped  organism; 
when  situated  at  the  end,  a  u  Trommelschlager,  "  or  drum- 
stick-shaped one.  As  the  degeneration  of  the  protoplasm 
of  the  bacillus  sets  the  spore  free,  it  appears  as  a  clear, 


BACTERIA.  35 

highly- refracting  sphere  or  ovoid  situated  in  a  little  col- 
lection of  granular  matter. 

Spores  differ  from  the  bacteria  in  that  their  capsules 
seem  to  prevent  evaporation  and  to  enable  them  to  with- 
stand drying  and  the  application  of  a  considerable  amount 
of  heat.  Ordinarily,  bacteria  are  unable  to  resist  a  tem- 
perature above  60°  C.  for  any  considerable  length  of 
time,  only  a  few  resistant  forms  tolerating  a  temperature 
of  70°  C.  The  spores,  however,  are  uninjured  by  such 
temperatures,  and  can  even  successfully  resist  that  of 
boiling  water  (100°  C.)  for  a  short  time.  The  extreme 
desiccation  caused  by  a  protracted  exposure  to  a  tem- 
perature of  150°  C.  will,  however,  destroy  them.  Not  only 
can  the  spores  resist  a  considerable  degree  of  heat,  but 
they  are  also  unaffected  by  cold  of  almost  any  intensity. 

While  the  cell-wall  of  the  bacterium  is  easily  pene- 
trated by  solutions  of  the  anilin  dyes,  it  is  a  matter  of 
much  difficulty  to  accomplish  the  staining  of  spores,  so 
that  we  see  they  are  probably  more  resistant  to  the 
action  of  chemical  agents  than  the  bacteria  themselves. 

When  a  spore  is  accidentally  dropped  into  some  nu- 
trient medium  a  change  is  shortly  observed.  The  proto- 
plasm, which  has  been  clear,  becomes  somewhat  granu- 
lar, the  capsule  a  little  less  distinct;  the  body  increases 
slightly  in  size,  and  in  the  course  of  time  splits  open  to 
allow  the  escape  of  the  young  organism.  The  direction 
in  which  the  escape  of  the  young  bacillus  takes  place  is 
of  interest,  as  varying  in  the  different  species.  The 
Bacillus  subtilis  escapes  from  the  end  of  the  spore,  where 
a  longitudinal  fissure  occurs;  the  bacillus  of  anthrax 
escapes  from  the  side,  sometimes  leaving  the  capsule  of 
the  spore  in  the  shape  of  two  small  cups. 

As  soon  as  the  young  bacillus  escapes  it  begins  to  in- 
crease in  size,  develops  around  its  soft  protoplasm  a  cha- 
racteristic capsule,  and,  having  once  established  itself, 
presently  begins  the  propagation  of  its  species  by  fission. 

In  addition  to  the  endospores,  of  which  we  have  just 
been  speaking,  there  are  arthrospores.  The  formation 


36  PATHOGENIC  BACTERIA. 

of  these  is  much  less  clear.  It  seems  to  be  an  effort  to 
convert  the  entire  microbe  into  a  permanent  form.  This 
process  is  observed  particularly  in  the  micrococci,  where 
the  substance  of  a  cell  is  said  to  break  up  into  segments, 
each  of  which  becomes  a  resisting  body  fruitful  in  prop- 
agating its  species.  Of  the  arthrospores  little  has,  so 
far,  been  learned.  It  is  not  improbable  that  among  the 
micrococci,  and  also  among  some  of  the  smaller  bacilli 
in  whom  no  spores  have  been  observed,  the  maintenance 
of  the  species  when  conditions  of  life  become  unfavor- 
able is  due  to  the  assumption  of  a  permanent  form  by 
some  of  the  individuals,  without  the  formation  of  any 
spore-like  bodies.  This  is  at  present  largely  a  matter  of 
conjecture,  but  the  indications  pointing  in  that  direction 
are  numerous. 

It  is  believed  by  Frankel  and  others  that  sporulation 
in  the  bacteria  is  not  a  sign  of  the  exhaustion  of  nutri- 
tion, but  a  sign  of  the  vital  perfection  of  the  organism. 
These  observers  regard  spore-formation  as  analogous  to 
the  flowering  of  higher  plants,  which  takes  place  only 
when  the  conditions  and  development  are  best. 

Morphology. — The  morphology  of  the  bacteria  is  quite 
varied.  Three  principal  forms,  however,  exist,  from  which 
the  others  seem  to  be  but  variations. 

The  most  simple  appear  as  minute  spheres,  and  from 


g 

© 


FIG.  2.  —  Diagram  illustrating  the  morphology  of  the  cocci  :  a,  coccus  or 
micrococcus  ;  6,  diplococcus  ;  t,  d,  streptococci  ;  e,  f,  tetragenococci  or  meris- 
mopedia  ;  g,  h,  modes  of  division  of  cocci  ;  i,  sarcina  ;  j,  coccus  with  flagella  ; 
k>  staphylococci. 

their  fancied  resemblance  to  little  berries  are  called  cocci 
or  micrococci  (Fig.  2,  a).     When  the  bacteria  of  this  form 


BACTERIA.  37 

multiply  by  fission  the  resulting  two  organisms  not 
infrequently  remain  attached  to  each  other,  producing 
what  is  called  a  diplococctis  (Fig.  2,  b).  The  diplococci 
sometimes  consist  of  two  perfect  spheres,  but  more  often 
show  a  flattening  of  the  contiguous  surfaces,  which  are 
not  in  absolute  apposition  (Fig.  2,  g).  In  a  few  cases,  as 
the  gonococcus,  the  approximated  surfaces  are  slightly 
concave,  causing  the  organism  to  somewhat  resemble  the 
German  biscuit  called  a  "semmel,"  hence  biscuit-  or 
semmel-cocci  (Fig.  2,  /z).  Frequently  a  second  binary  di- 
vision occurs,  causing  four  individuals  to  remain  closely 
approximated,  without  disturbing  the  arrangement  of  the 
first  two.  When  division  of  this  kind  produces  a  distinct 
tetrad,  the  organism  is  described  as  a  tetragenococcus, 
while  to  the  entire  class  of  cocci  dividing  so  as  to  pro- 
duce fours,  eights,  twelves,  etc.  on  the  same  plane  the 
name  merismopedia  is  given  (Fig.  2,  e  and  f). 

If,  as  sometimes  happens,  the  divisions  take  place  in 
three  directions,  so  as  to  produce  cubical  masses  or  "pack- 
ages" of  cocci,  the  resulting  aggregation  is  described  as 
a  sarcina  (Fig.  2,  i).  This  form  slightly  resembles  a  dice 
or  a  bale  of  cotton  in  miniature. 

If  the  divisions  always  take  place  in  the  same  direc- 
tion, so  as  to  produce  a  chain  or  string  of  beads,  the 
organism  is  described  as  streptococcus  (Fig.  2,  d\  When 
there  are  diplococci  joined  in  this  manner  a  strepto-diplo- 
coccus  is  of  course  formed. 

More  common  than  any  of  the  forms  already  described 
is  one  in  which,  without  any  definite  arrangement,  the 
cocci  occur  in  irregular  groups  having  a  fancied  resem- 
blance to  bunches  of  grapes.  These  are  called  staphylo- 
cocci,  and,  as  it  is  very  unusual  to  find  cocci  habitually 
occurring  isolated,  most  cocci  not  classified  under  one  of 
the  above  heads  are  called  staphylococci. 

When  cocci  are  associated  in  globular  or  lobulated 
clusters  encased  in  a  resisting  glutinous,  homogeneous 
mass,  the  name  ascococcus  has  been  used  in  describing 
them.  A  modified  form  of  this,  in  which  the  cocci  are 


38  PATHOGENIC  BACTERIA. 

in  chains  or  solitary  and  are  surrounded  by  an  encase- 
ment almost  cartilaginous  in  consistence,  has  been  called 
leuconostoc. 

Certain  bacteria,  constituting  a  better-known  if  not 
more  important  group,  are  not  spherical,  but  elongate 
or  "rod-shaped,"  and  bear  the  name  bacillus  (Fig.  3). 


FIG.  3. — Diagram  illustrating  the  morphology  of  the  bacilli :  a,  b,  c,  various 
forms  of  bacilli ;  d,  e,  bacilli  with  flagella ;  f,  chain  of  bacilli,  individuals  dis- 
tinct ;  g,  chain  of  bacilli,  individuals  not  separated. 

I  would  remark  that  the  absence  of  a  standard  by 
which  to  separate  the  cocci  from  the  bacilli  is  the  cause 
of  much  confusion.  In  the  judgment  of  the  author,  it 
would  be  well  to  place  all  individuals  having  one  diam- 
eter greater  than  the  other  among  the  bacilli.  This 
would  prevent  the  error  of  describing  one  species  as 
"oval  cocci"  and  another  as  "nearly  round  bacilli," 
and  by  giving  a  definite  standard  would  greatly  aid  in 
the  formation  of  a  differential  table. 

The  bacilli  present  a  considerable  variety  of  forms. 
Some  are  quite  short,  with  rounded  ends,  so  as  to  ap- 
pear elliptical  ;  some  are  long  and  delicate.  Some  have 
rounded  ends,  as  subtilis  ;  others  have  square  ends,  as 
anthrax.  Some  are  enormously  large,  some  exceedingly 
small.  Some  are  always  isolated,  never  forming  threads 
or  chains  ;  others  nearly  always  occur  in  these  forms. 

The  bacilli  always  divide  by  transverse  fission,  so  that 
the  only  peculiarity  of  arrangement  is  the  formation  of 
threads  or  chains. 

In  the  older  writings  the  short,  stout  bacilli  were  all 
described  under  the  generic  term  bacterium.  This  genus, 
like  some  of  the  species  it  comprehended,  has  now  passed 


BACTERIA.  39 

out  of  use.  Some  of  the  flexile  bacilli,  whose  movements 
are  sinuous,  much  resembling  the  swimming  of  a  snake 
or  an  eel,  were  described  as  vibrio,  but  this  name  also  has 
passed  into  disuse. 

The  long  filaments  formed  by  the  division  of  bacilli 
without  their  distinct  separation  are  sometimes  called 
leptothrix,  and  when  these  long  threads  form  distinct 
masses  surrounded  by  a  jelly-like  material,  the  name 
myconostoc  is  sometimes  applied  to  them. 

Certain  forms  much  resembling  bacilli  in  their  isolated 
state,  characterized  by  the  formation  of  long  filaments 
with  a  peculiar  grouping  which  gives  the  appearance 
of  a  false  branching,  are  described  as  cladothrix ;  others 
in  which  true  branchings  are  seen,  as  streptothrix.  One 
other  bacillus-like  form,  consisting  of  long,  thick,  not 
distinctly  segmented,  straight  threads,  is  called  beggiatoa. 
The  only  important  difference  between  it  and  leptothrix 
is  that  its  filaments  are  thick  and  coarse,  while  those  of 
leptothrix  are  very  delicate. 

Some  of  the  elongate  bacteria  have  a  remarkably 
twisted  form  and  bear  some  resemblance  to  a  cork- 
screw. These  are  called  spirilla  (Fig.  4).  A  subdivision 


FIG.  4. — Diagram  illustrating  the  morphology  of  the  spirilla :  a,  6,  c,  spirilla ; 
d,  e,  spirochaeta. 

of  them,  whose  individuals  are  not  only  twisted  but  are 
also  very  flexible,  is  called  spiroduzta.  Though  not 
formerly  differentiated  from  vibrio,  these  forms  are  quite 
distinct. 

A  spiral  organism  of  a  ribbon  shape  is  called  spiro- 


40  PATHOGENIC  BACTERIA. 

monas,  while  a  similar  organism  of  spindle  shape  is 
called  a  spirulina.  One  species  of  spiral  bacteria  in 
whose  protoplasm  sulphur-grounds  have  been  detected 
has  been  called  ophidiomonas. 

Some  of  the  spirilla  are  exceedingly  long  and  deli- 
cate, as  the  spirochaeta  of  relapsing  fever ;  others  which 
are  stouter,  like  the  spirillum  of  cholera,  habitually  occur 
in  such  short  individuals  as  to  be  easily  mistaken  for 
slightly-bent  bacilli. 

Classification. — Leeuwenhoek,  when  he  first  saw  the 
bacteria — and  his  successors  even  to  so  recent  a  date  as 
to  include  Khrenberg  and  Dujardin — did  not  doubt  that 
they  belonged  to  the  infusoria. 

It  was  not  until  biologists  had  concluded  that  organ- 
isms which  take  into  their  bodies  particles  of  solid  or 
semi-solid  material,  digest  that  which  is  useful,  and 
extrude  the  remainder,  are  animals,  and  that  those  which 
live  purely  by  osmosis  and  exosmosis  are  vegetables,  that 
the  bacteria,  which  we  have  seen  provided  with  a  resist- 
ant cell-wall,  allowing  of  no  possibility  of  nutrition 
except  by  osmosis  and  exosmosis,  could  be  finally  and 
correctly  classed  among  the  members  of  the  vegetable 
kingdom. 

The  extremely  simple  organization  of  bacteria  naturally 
places  them  among  the  lowest  members  of  the  vegetable 
kingdom,  in  that  class  of  the  Cryptogamia  known  as 
Thallophytae,  comprising  the  algae,  lichens,  and  fungi. 

The  algae  are  mostly  water-plants,  containing  chloro- 
phyl  and  obtaining  their  nourishment  from  inorganic 
substances. 

The  lichens  are  plants,  some  of  which  contain  chloro- 
phyl.  They  live  upon  inorganic  matter,  which  they 
generally  absorb  from  the  air.  According  to  the  new 
view  of  the  subject,  some,  if  not  all,  of  these  plants  are 
regarded  as  fungi  growing  parasitically  upon  algae. 

The  fungi,  the  lowest  group  of  all,  are  minute  or  large 
plants,  mostly  devoid  of  chlorophyl,  living  upon  organic 
matter,  which  they  obtain  as  saprophytes  from  decom- 


BACTERIA.  41 

posing  animal   and   vegetable   matters,   or  as   parasites 
apon  the  tissues  or  juices  of  living  animals  or  plants. 
This  lowest  family,  the  fungi,  are  divisible  into  the — 

Hyphomycetes  or  Mucorini,  or  moulds; 
Saccharomycetes,  or  yeasts;  and 
Schizomycetes,  or  bacteria. 

Cohn  divided  the  bacteria,  according  to  their  mor- 
phology, into — 

Sphero-bacteria,  or  cocci ; 
Micro-bacteria — short  rods  ; 
Desmo-bacteria — bacilli ; 
Spiro-bacteria — spirilla. 

Davaine  suggested  a  classification  based  upon  motility, 
making  four  classes — Bacterium,  Vibrio,  Bacteridium, 
and  Spirillum,  neglecting  to  provide  for  the  cocci. 

Zoph  arranged  them,  according  to  his  theory  of 
pleomorphism,  into  the  COCCACE^E,  comprising  those 
known  only  in  the  coccus  form,  and  comprehending 
the  streptococci^  merismopedia,  sarcina,  micrococcus,  and 
ascococcus ;  the  BACTERIACE^E,  comprehending  the  genera 
bacterium,  spirillum,  vibrio,  leuconostoc,  bacillus,  and 
clostridium  (chiefly  coccus,  rod,  and  thread  forms  ;  the 
former  may  be  absent ;  in  the  latter  there  is  no  distinction 
between  base  and  apex  ;  threads  straight  or  screw-like)  ; 
and  the  L,EPTOTHRICHE^E,  comprehending  crenothrix, 
beggiatoa,  phragmidiothrix,  and  leptothrix  (coccus,  rod, 
and  thread  forms  ;  the  latter  show  a  distinction  between 
base  and  apex  ;  threads  straight  or  screw-like  ;  spore- 
formation  not  demonstrated). 

This  classification  is,  however,  based  upon  what  is 
probably  an  erroneous  principle,  the  pleomorphism  of 
the  bacteria. 

Van  Tieghem,  DeBary,  and  Hiippe  formed  classifica- 
tions the  main  feature  of  which  was  the  formation  of 
endospores  or  arthrospores,  but,  as  the  sporulation  of 
many  species  is  as  yet  unknown,  they  cannot  be  properly 
placed  in  it. 


42  PATHOGENIC  BACTERIA. 

It  has  even  been  suggested  to  classify  the  bacteria  by 
the  size  and  number  of  their  flagella,  of  which  so  little 
is  known. 

The  most  convenient  classification,  though  it  cannot 
be  purely  scientific,  seems  to  be  the  morphological  one 
given  by  Cohn.  Baumgarten,  recognizing  the  relative 
pleomorphism  of  certain  of  the  species,  has  modified  it 
as  follows,  and  thus  made  it  answer  all  the  needs  of  the 
pathologist  at  least: 

I.  Cocci,  \ 

II.  Bacilli,  i-  species  relatively  monomorphous. 

III.  Spirilla,  J 

IV.  Spirulina,  \ 

V.  Leptothrix,  I  species  relatively  pleomorphous. 
VI.  Cladothrix,) 

The  members  of  the  first  group,  the  cocci,  bacilli,  and 
spirilla,  are  practically  the  only  ones  which  are  of  patho- 
logical significance. 


CHAPTER  II. 
BIOLOGY   OF  BACTERIA. 

THE  distribution  of  bacteria  is  wellnigh  universal. 
They  and  their  spores  float  in  the  atmosphere  we  breathe, 
swim  in  the  water  we  drink,  grow  upon  the  food  we  eat, 
and  luxuriate  in  the  soil  beneath  our  feet.  Nor  is  this 
all,  for,  entering  the  palpebral  fissures,  they  develop  upon 
the  conjunctiva  ;  entering  the  nares,  they  establish  them- 
selves in  the  nose  ;  the  mouth  is  always  replete  with 
them  ;  and,  as  many  are  swallowed,  the  digestive  appa- 
ratus always  contains  them.  The  surface  of  the  body 
never  escapes  their  establishment,  and  so  deeply  are 
some  individuals  situated  beneath  the  epithelial  cells 
that  the  most  careful  washing  and  scrubbing  and  the  use 
of  the  most  powerful  germicides  are  required  to  rid  the 
surgeon's  hands  of  what  may  prove  to  be  dangerous 
hindrances  to  the  healing  of  wounds.  The  ear  is  not 
without  its  microscopic  flora  ;  special  varieties  live  be- 
neath the  finger-nails,  and  especially  the  toe-nails,  in 
the  vagina,  and  beneath  the  prepuce. 

While  so  general,  however,  they  are  not  ubiquitous. 
Tyndall  succeeded  in  proving  that  the  atmosphere  of 
high  Alpine  altitudes  was  free  from  them,  and  likewise 
that  the  glacier  ice  contained  none.  Wherever  man,  ani- 
mals, or  even  plants,  live,  die,  and  decompose,  bacteria 
are  sure  to  be  present. 

Notwithstanding  their  extreme  familiarity  with  the 
animal  body,  there  are  certain  parts  of  it  into  which 
bacteria  do  not  enter,  for  the  body-juices  and  tissues  of 
normal  animals  are  constantly  free  from  them,  and  their 
occurrence  there  may  be  accepted  as  a  sign  of  disease. 

The   presence  of  bacteria  in  the  air  is  generally  de- 

43 


44  PA  THOGENIC  BA  CTERIA . 

pendent  upon  their  previous  existence  in  the  soil,  its  pul- 
verization, and  its  distribution  by  currents  of  the  atmo- 
sphere. Koch  has  shown  that  the  upper  stratum  of  the 
soil  is  exceedingly  rich  in  bacteria,  but  that  their  num- 
bers decrease  as  the  soil  is  penetrated,  until  below  a 
depth  of  one  meter  there  are  very  few.  Remembering 
that  bacteria  can  live  only  upon  organic  matter,  this  is 
readily  understandable.  Most  of  the  organic  matter  is 
upon  the  surface  of  the  soil.  Where,  as  in  the  case  of 
porous  soil  or  the  presence  of  cesspools  and  dung-heaps, 
the  decomposing  materials  are  allowed  to  penetrate  to  a 
considerable  depth,  the  bacteria  may  occur  much  farther 
from  the  surface,  yet  they  are  rarely  found  at  any  great 
depth,  because  the  majority  of  the  known  species  require 
oxygen. 

The  water  of  stagnant  pools  always  teems  with  bacte- 
ria, but  that  of  deep  wells  rarely  contains  many  unless 
it. is  polluted  from  the  surface  of  the  earth. 

Being  generally  present  in  the  soil,  which  the  feet  of 
men  and  animals  grind  to  powder,  the  bacteria,  together 
with  the  pulverized  earth,  are  blown  from  place  to  place 
into  every  nook  and  cranny,  until  it  is  impossible  to  es- 
cape them.  It  has  been  suggested  by  Soyka  that  the 
currents  of  air  passing  over  the  surface  of  liquids  might 
take  up  bacteria,  but,  although  he  seemed  to  show  it  ex- 
perimentally, it  is  not  generally  believed.  Where  bac- 
teria are  growing  in  colonies  they  seem  to  remain  un- 
disturbed by  currents  of  air  unless  the  surface  becomes 
roughened  or  broken. 

Most  of  the  bacteria  which  are  carried  about  by  the  air 
are  what  are  called  saprophytes,  and  are  perfectly  harm- 
less to  the  human  being  ;  but  not  all  belong  to  this  class, 
nor  will  they  do  so  while  tuberculous  patients  are  al- 
lowed to  expectorate  upon  the  sidewalks,  and  typhoid 
patients'  wash  to  dry  upon  the  clothes-line,  and  their 
dejecta  to  be  spread  upon  the  ground. 

The  growth  of  bacteria  is  profoundly  influenced  by 
environment,  so  that  a  consideration  of  the  conditions 


BIOLOGY  OF  BACTERIA.  45 

favorable  or  detrimental  to  their  existence  becomes  a 
necessity. 

Conditions  influencing-  the  Growth  of  Bacteria. — 
(a)  Oxygen. — The  majority  of  bacteria  grow  best  when 
exposed  to  the  air.  Some  develop  better  when  the  air  is 
withheld;  some  will  not  grow  at  all  where  the  least 
amount  of  oxygen  is  present.  Because  of  these  pecu- 
liarities bacteria  are  divisible  into  the 

Aerobic  bacteria,  those  growing  in  oxygen. 

Anaerobic  bacteria,  those  not  growing  in  the  presence 
of  oxygen. 

As,  however,  some  of  the  aerobic  forms  will  grow 
almost  as  well  without  as  with  oxygen,  the  term  optional 
(facultative)  anaerobics  has  been  applied  to  the  special 
class  made  to  include  them. 

As  examples  of  strictly  aerobic  bacteria  the  Bacillus 
subtilis  and  the  Bacillus  aerophilus  may  be  given.  These 
forms  will  not  grow  if  oxygen  is  denied  them.  The 
staphylococci  of  suppuration  and  the  bacilli  of  typhoid 
fever,  pneumonia,  and  anthrax,  as  well  as  the  spirillum 
of  cholera,  will  grow  almost  equally  well  with  or  with- 
out oxygen,  and  hence  belong  to  the  optional  anaerobics. 
The  bacillus  of  tetanus  and  of  malignant  edema,  and  the 
non-pathogenic  forms,  the  Bacillus  butyricus,  Bacillus 
muscoides,  and  Bacillus  polypiformis,  will  not  develop 
at  all  where  any  oxygen  is  present,  and  hence  are 
strictly  anaerobic. 

(b)  Nutriment. — The  bacteria  do  not  seem  able  to  derive 
their  nourishment  from  purely  inorganic  matter.  Pros- 
kauer  and  Beck,  however,  have  succeeded  in  growing  the 
tubercle  bacillus  in  a  mixture  containing  ammonium 
carbonate  0.35  per  cent,  potassium  phosphate  0.15  per 
cent,  magnesium  sulphate  0.25  per  cent,  glycerin  1.5 
per  cent  They  grow  best  where  diffusible  albumins  are 
present.  The  ammonium  salts  are  rather  less  fitted  to 
support  them  than  their  organic  compounds.  The  in- 
dividual bacterium  varies  very  widely  in  the  nutriment 
which  it  requires.  Some  of  the  water-microbes  can  live 


46  PATHOGENIC  BACTERIA. 

in  distilled  water  to  which  the  smallest  amount  of  organic 
matter  has  been  added;  others  require  so  concentrated  a 
medium  that  only  blood-serum  can  be  used  for  their 
cultivation.  Sometimes  a  species  with  a  preference  for  a 
particular  culture-medium  can  gradually  be  accustomed 
to  another,  though  immediate  transplantation  causes  the 
death  of  the  transplanted  organism.  Sometimes  the  ad- 
dition of  such  substances  as  glucose  and  glycerin  has  a 
peculiarly  favorable  influence  upon  bacteria,  causing,  for 
example,  the  tubercle  bacillus  to  grow  upon  agar-agar. 

(c)  Moisture. — A  certain  amount  of  water  is  always 
necessary  for  the  growth  of  bacteria.  The  amount  can 
be  exceedingly  small,  however,  so  that  the  Bacillus  pro- 
digiosus  is  able  to  develop  successfully  upon  crackers  and 
dried  bread.  Materials  used  as  culture-media  should  not 
be  too  concentrated;  at  least  80  per  cent,  of  water  should 
be-  present.  Most  bacteria  grow  best  in  liquid  media; 
that  is,  they  form  the  longest  threads,  and  diffuse  them- 
selves throughout  the  liquid  so  as  to  be  present  in  far 
greater  numbers  than  when  on  solid  media. 

The  statement  that  certain  forms  of  bacteria  can  flour- 
ish in  clean  distilled  water  seems  to  be  untrue.  When 
transferred  to  such  a  medium  the  organisms  soon  die  and 
undergo  a  granular  degeneration  of  their  substance.  If, 
however,  in  their  introduction  a.  good-sized  drop  of  cul- 
ture-material is  carried  with  them,  the  distilled  water 
ceases  to  be  such,  and  becomes  a  dilute  bouillon  fitted  to 
support  life  for  a  time. 

(cC)  Reaction. — Should  the  pabulum  supplied  to  bacte- 
ria contain  an  excess  of  either  alkali  or  acid,  the  growth 
of  the  organisms  is  inhibited.  Most  true  bacteria  grow 
best  in  a  neutral  or  feebly  alkaline  medium.  There  are 
exceptions  to  this  rule,  for  the  Bacillus  butyricus  and  the 
Sarcina  ventriculi  can  grow  well  in  strong  acids,  and  the 
Micrococcus  urea  can  tolerate  excessive  alkalinity.  Acid 
media  are  excellent  for  the  cultivation  of  moulds. 

(e)  Light. — Most  species  of  bacteria  are  not  influenced 
in  their  growth  by  the  presence  or  absence  of  light.  The 


BIOLOGY  OF  BACTERIA.  47 

direct  rays  of  the  sun,  and  to  a  less  degree  the  intense 
rays  of  the  electric  arc-light,  retard  and  in  numerous  in- 
stances kill  bacteria.  Some  colors  are  distinctly  inhibi- 
tory to  their  growth,  blue  being  especially  prejudicial. 
Some  of  the  chromogenic  forms  will  only  produce  their 
colors  when  exposed  to  the  ordinary  light  of  the  room. 
The  Bacillus  mycoides  roseus  will  not  produce  its  red 
pigment  except  in  the  absence  of  light.  The  pathogenic 
bacteria  have  their  virulence  gradually  attenuated  if 
grown  in  the  light. 

(f)  Electricity. — Very  little  is  known  about  the  action 
of  electric  currents  upon  bacteria.     Very  powerful  dis- 
charges of  electricity  through  culture-media  are  said  to 
kill  the  organisms. 

(g)  Movement. — When  bacteria  are  growing  in  a  liquid 
medium    perfect   rest   seems   to   be   the   condition   best 
adapted  for  their  development.     A  slow-flowing  move- 
ment does  not  have  much  inhibitory  action,  but  violent 
agitation,  as  by  shaking  a  culture  in  a  machine,  greatly 
hinders  or  prevents  their  growth.     The  practical  appli- 
cation of  this  will  show  that  rapidly-flowing  streams, 
whose  currents  are  interrupted  by  falls  and  rapids,  will, 
other  things  being  equal,  furnish  a  better  drinking-water 
than  a  deep,  still-flowing  river. 

(ti)  Temperature. — The  question  of  temperature  is  of 
importance  from  its  bearing  upon  sterilization.  Accord- 
ing to  Frankel,  bacteria  will  scarcely  grow  at  all  below 
1 6°  and  above  40°  C. 

The  researches  of  Fliigge  show  that  the  Bacillus  sub- 
tilis  will  grow  very  slowly  at  6°  C. ,  and  as  the  tempera- 
ture is  elevated  it  is  said  that  until  12.5°  C.  is  reached 
fission  does  not  occur  oftener  than  every  four  or  five 
hours.  When  25°  C.  is  reached  the  fission  occurs  every 
three-quarters  of  an  hour,  and  at  30°  C.  about  every  half 
hour. 

Most  bacteria  die  at  a  higher  temperature  than  60- 
75°  C.  The  spores  can  resist  boiling  water,  but  are 
killed  by  dry  heat  if  exposed  to  150°  C.  for  an  hour  or  to 


48  PATHOGENIC  BACTERIA. 

175°  C.  for  five  to  ten  minutes.  Freezing  kills  many,  but 
not  all  bacteria,  but  does  not  affect  the  spores  at  all. 

Most  bacteria  grow  best  at  the  ordinary  temperature  of 
a  comfortably  heated  room,  and  are  not  affected  by  its 
occasional  slight  changes.  Some,  chiefly  the  pathogenic 
forms,  are  not  cultivable  except  at  the  temperature  of 
the  animal  body  (37°  C.) ;  others,  like  the  tubercle  bacil- 
lus, grow  best  at  a  temperature  a  little  above  that  of  the 
body— 40°  C. 

Some  forms  of  the  bacteria  are  never  found  except  in 
the  tissues  of  diseased  animals.  Such  organisms  are 
called  parasites.  The  parasitic  group  really  is  divisible 
into  the  purely  parasitic  and  the  occasionally  parasitic 
bacteria.  Of  the  first  division  the  tubercle  bacillus  may 
be  used  as  an  illustration,  for,  so  far  as  is  known,  it  is 
never  found  in  other  places  than  the  bodies  and  dejecta 
of  diseased  animals.  The  cholera  spirillum  illustrates 
the  second  group,  for,  while  it  produces  the  disease 
known  as  Asiatic  cholera  when  admitted  to  the  digestive 
tract,  it  is  a  constant  inhabitant  of  certain  waters,  where 
it  multiplies  with  luxuriance. 

Bacteria  which  do  not  enter  the  animal  economy,  or  if 
accidentally  admitted  do  no  harm,  but  live  upon  decaying 
animal  and  vegetable  materials,  are  called  saprophytes. 
The  parasitic  organisms  alone  possess  much  interest  to 
the  physician,  but  as  in  their  growth  the  saprophytes  ex- 
hibit many  interesting  vital  manifestations,  it  is  not  well 
to  exclude  them  or  their  products  from  the  following 
consideration  of  the 

Results  of  Vital  Activity  in  Bacteria. — i.  Fermenta- 
tion.— The  alcoholic  fermentation,  which  is  a  familiar  phe- 
nomenon to  the  layman  as  well  as  to  the  brewer  and  the 
chemist,  is  not  the  work  of  a  bacterium,  but  of  a  yeast- 
plant,  one  of  the  saccharomyces  fungi.  The  acetic-acid, 
lactic-acid,  and  butyric-acid  fermentations  are,  however, 
caused  by  bacilli.  A  considerable  number  of  bacilli  seem 
capable  of  converting  milk-sugar  into  lactic  acid,  some- 
times associating  this  with  coagulation  of  milk,  some- 


BIOLOGY  OF  BACTERIA.  49 

times  not.  The  production  of  coagulation  in  milk  is  not 
always  associated  with  acid-production,  but  with  the  pro- 
duction of  a  curdling  ferment  similar  to  that  belonging 
to  the  gastric  juice.  There  seems  to  be  no  real  specific 
micro-organism  for  the  lactic-acid  fermentation,  although 
the  Bacillus  acidi  lactici  seems  to  be  the  most  powerful 
generator  of  the  acid.  There  may  also  be  several  bac- 
teria which  produce  the  acetic  fermentation,  though  it  is 
generally  attributed  to  a  special  common  form,  the  Myco- 
derma  aceti  or  Bacillus  aceticus.  The  butyric  fermenta- 
tion is  generally  due  to  the  Bacillus  butyricus,  though  it 
also  may  be  caused  by  other  bacilli,  the  one  named  sim- 
ply being  the  most  common.  (For  an  exact  description 
of  the  chemistry  of  the  fermentations  reference  must  be 
made  to  text-books  upon  that  subject,  as  their  considera- 
tion here  would  occupy  too  much  space.) 

2.  Putrefaction. — This  process  is  in  many  respects  sim- 
ilar to  the  preceding,  except  that  instead  of  occurring  in 
carbohydrates  it  takes  place  in  nitrogenous  bodies.  The 
first  step  seems  to  be  the  transformation  of  the  albumins 
into  peptones,  then  the  splitting  up  of  the  peptones  into 
a  large  number  of  gases,  acids,  bases,  and  salts.  In  the 
process  the  innocuous  albumins  are  frequently  changed  to 
toxalbumins,  and  sometimes  to  distinct  animal  alkaloids 
known  as  ptomaines.  Vaughan  and  Novy  declare  the 
term  "animal  alkaloid"  to  be  a  misnomer,  as  ptomaines 
are  sometimes  produced  from  vegetable  substances  in 
the  process  of  decomposition ;  they  suggest  the  term 
u putrefactive  alkaloids"  as  preferable.  The  definition 
of  a  ptomaine  given  by  these  observers  is  ua  chemical 
compound,  basic  in  character,  formed  by  the  action  of 
bacteria  on  organic  matter."  The  chemistry  of  these 
bodies  is  very  complex,  and  for  a  satisfactory  description 
of  them  Vaughan  and  Novy's  book1  is  brief  and  excel- 
lent. Among  the  ptomaines  the  following  appear  to 
be  important:  Methylamin  (CH3NH2),  the  simplest  or- 
ganic base  formed  in  the  process  of  putrefaction;  dime- 

1  Ptomaines  and  Leucomalnes. 
4 


50  PATHOGENIC  BACTERIA. 

thylamin  ((CH3)2NH) ;  trimethylamin  (C3H9N  =  (CH3)3N) ; 
ethylamin  (C2H5.NH2);  diethylamin  (C4HnN  =  (C2H5)2- 
NH) ;  triethylamin  (C6H15N  =  (C2H5)3N) ;  propylamin 
(C3H7.NH2) ;  butylamin  C4HUN) ;  iso-amylamin  ;  caproyl- 
amin  ;  tetanotoxin  ;  spasmotoxin  ;  dihydrolutidin  ;  putres- 
cin  ;  cadaverin  ;  neuridin  ;  saprin  ;  pyocyanin  ;  and  tyro- 
toxicon.  Numerous  others  have  been  described,  some 
toxic,  others  harmless. 

3.  Chr ontogenesis. — Those  bacteria  which  produce  col- 
ored colonies  or  impart  color  to  the  medium  in  which 
they  grow  are  called  chromogenic ;  those  with  which  no 
color  is  associated,  non-chromogenic.  Most  chromogenic 
bacteria  are  saprophytic  and  non-pathogenic.  Some  of 
the  pathogenic  forms,  as  the  Staphylococcus  pyogenes 
aureus  and  citreus,  are,  however,  color-producers.  It 
seems  likely  that  the  bacteria  do  not  form  the  actual 
pigments,  but  certain  chromogenetic  substances  which, 
uniting  with  substances  in  the  culture-medium,  pro- 
duce the  colors. 

Galleotti  has  described  two  kinds  of  pigment,  one  of 
which,  being  soluble,  readily  penetrates  all  neighboring 
portions  of  the  culture-medium,  like  the  colors  of  Bacillus 
pyocyaneus,  and  an  insoluble  pigment  which  does  not 
tinge  the  solid  culture-media  at  all,  but  is  constantly 
found  associated  with  the  colonies,  like  the  pigment  of 
Bacillus  prodigiosus.  The  pigments  are  found  in  their 
greatest  intensity  near  the  surface  of  the  colony.  The 
coloring  matter  never  occupies  the  protoplasm  of  the 
bacteria  (except  the  Bacillus  prodigiosus,  in  whose  cells 
occasional  pigment-granules  may  be  seen),  but  occurs  in 
an  intercellular  excrementitious  substance. 

The  pigments  are  so  varied  as  to  give  almost  every 
known  color.  It  sometimes  happens  that  a  bacterium 
will  elaborate  two  or  more  colors.  The  Bacillus  pyo- 
cyaneus thus  produces  pyocyanin  and  fluorescin,  both 
being  soluble  pigments — one  blue,  the  other  green. 
Gessard  has  shown  that  when  the  Bacillus  pyocyaneus 
is  cultivated  upon  white  of  egg,  it  produces  only  the 


UNIVk-RSITY 

OF 


BIOLOGY  OF  BACTERIA.  51 

green  fluorescent  pigment,  while  in  pure  peptone  solu- 
tion it  grows  with  the  production  of  blue  pyocyanin 
alone.  His  experiments  prove  a  very  interesting  fact, 
that  for  the  production  of  fluorescin  it  is  necessary  that 
the  culture-medium  contain  a  definite  amount  of  a 
phosphatic  salt.  Sometimes  one  pigment  is  soluble, 
the  other  insoluble,  so  that  the  colony  will  appear  one 
color,  the  medium  upon  which  it  grows  another.  Some 
organisms  will  only  produce  their  colors  in  the  light ; 
others,  as  the  Bacillus  mycoides  roseus,  only  in  the  dark. 
Some  produce  them  only  at  the  room- temperature,  but, 
though  growing  luxuriantly  in  the  incubator,  refuse  to 
produce  pigments  at  so  high  a  temperature.  Thus, 
Bacillus  prodigiosus  produces  a  brilliant  red  color  when 
growing  at  the  temperature  of  the  room,  but  is  colorless 
when  grown  in  the  incubator.  Colored  lights  seem  to 
have  no  modifying  influence  upon  the  pigment-produc- 
tion. Even  if  for  successive  generations  the  bacterium 
be  grown  so  as  to  be  colorless,  it  speedily  recovers  its 
primitive  color  when  restored  to  its  old  environment,  no 
matter  what  the  color  of  the  light  thrown  upon  it.  Bac- 
teria which  have  been  robbed  of  their  color  by  incuba- 
tion, when  placed  in  the  normal  environment  produce 
the  original  color,  no  matter  what  color  the  light  they 
receive.  Some  of  the  pigments — perhaps  most  of  them — 
are  formed  only  in  the  presence  of  oxygen. 

4.  Liquefaction  of  Gelatin. — When  certain  forms  of 
bacteria  are  grown  in  gelatin  the  culture-medium  is 
partly  or  entirely  liquefied.  This  characteristic  is  en- 
tirely independent  of  any  other  property  of  the  bacte- 
rium, and  is  one  manifested  alike  by  pathogenic  and 
non-pathogenic  individuals.  Sternberg  and  Bitter  have 
shown  that  if  from  a  culture  in  which  liquefaction  has 
taken  place  the  bacteria  be  removed  by  filtration,  the 
filtrate  will  retain  the  power  of  liquefying  gelatin,  show- 
ing that  the  property  is  not  resident  in  the  bacteria,  but 
in  some  substance  in  solution  in  their  excreted  products. 
These  products  are  described  as  utryptic  enzymes"  by 


52  PATHOGENIC  BACTERIA. 

Fermi,  who  found  that  heat  destroyed  them.  Mineral 
acids  seem  to  check  their  power  to  act  upon  gelatin. 
Formalin  renders  the  gelatin  insoluble.  As  some  of 
the  bacteria  not  only  liquefy  the  gelatin,  but  do  so  in  a 
peculiar  and  constantly  similar  manner,  the  presence  or 
absence  of  the  change  becomes  extremely  useful  for  the 
separation  of  different  species. 

5.  Production  of  Acids  and  Alkalies. — Under  the  head 
of  u  Fermentation "  the  formation  of  acetic,  lactic,  and 
butyric  acids  has  been  discussed.     These,  however,  are 
by  no  means  all  the  acids  resulting  from  microbic  me- 
tabolism.    Ziegler  mentions  formic,  propionic,  baldrianic, 
palmitic,  and  margaric  as  being  among  those  produced, 
and  even  this  list  may  not  comprehend  them  all.     As 
the  acidity  due  to  the  microbic  metabolism  progresses,  it 
impedes,  and  ultimately  completely  inhibits,  the  develop- 
ment of  the  bacteria.     The  addition  of  litmus  to  the  cul- 
ture-medium is  one  of  the  best  methods  for  detecting  the 
acids.     Milk  to  which  litmus  is  added  is  particularly  con- 
venient.    Rosalie  acid  may  also  be  used,  the  acid  convert- 
ing its  red  into  an  orange  color.     The  same  tests  will  also 
determine  the  alkali-production,  which  occurs  rather  less 
frequently  than  acid-formation,  and  depends  chiefly  upon 
the  salts  of  ammonium. 

6.  Production  of  Gases. — This  seems,  in  reality,  to  be 
a  part  of  the  process  of  decomposition  and  fermentation. 
Among  the  gases  due  to  bacterial  action,  CO2,  H2S,  NH4, 
CH4,  and  others  have  been  described.     If  the  bacterium 
be  anaerobic  and  develop  at  the  lower  part  of  a  tube  of 
gelatin,  not  infrequently  a  bubble  of  gas  will  be  formed 
about  the  colonies.     This  is  almost  constant  in  tetanus 
and   malignant   edema.      Ordinarily,   the  production  or 
liberation  of  gases  passes  undetected,  the  vapors  escaping 
from  the  surface  of  the  culture-medium. 

7.  Production  of  Odors. — Of  course  such  gases  as  H2S 
and  NH3  are  sufficiently  characteristic  to  be  described  as 
odors.     There  are,  however,  a  considerable   number  of 
pungent  odors  which  seem  dependent  purely  upon  odor- 


BIOLOGY  OF  BACTERIA.  53 

iferous  principles  dissociated  from  gases.  Many  of  the 
odors  are  extremely  unpleasant,  as  the  fetid  one  caused 
by  Bacillus  pyogenes  foetidus.  The  odor  does  not  have 
any  direct  relation  to  decomposition,  but,  like  the  colors 
and  acids,  seems  to  be  a  peculiar  individual  characteristic 
of  the  metaboiism  of  the  organism. 

8.  Production   of  Phosphorescence. — A  Bacillus   phos- 
phorescens  and  numerous  other   organisms   have  a  dis- 
tinct phosphorescence  associated  with  their  growth.     It 
is  said  that  so  much  illumination  is  sometimes  caused  by 
a  gelatin  culture  of  some  of  these  as  to  enable  one  to  tell 
the  time  by  a  watch.     Most  of  them  are  found  in  sea- 
water,  and  are  best  grown  in  sea- water  gelatin. 

9.  Production  of  Aromatic s. — The  most  important  of 
these  is  indol,  which  was  at  one  time  thought  to  be  pecu- 
liar to  the  cholera  spirillum.     At  present  we  know  that  a 
variety  of  organisms  produce  it,  and  that  it  and  phenol, 
kresol,  hydrochinon,  hydroparacumaric  acid,  and  paroxy- 
phenylic-acetic  acid  are  by  no  means  uncommon. 

10.  Reduction  of  Nitrites. — A  considerable  number  of 
bacteria  are  able  to  reduce  nitrites  present  in  the  soil  or 
in  culture-media  prepared  for  them  into  ammonia  and 
nitrogen.     To  the  horticulturist  this  is  a  matter  of  much 
interest.     Winogradsky  has   found  a  specific   nitrifying 
bacillus  in  soil,  and  asserts  that  the  presence  of  ordinary 
bacteria  in  the  soil  causes  the  reduction  of  no  nitrites  so 
long  as  his  special  bacillus  is  withheld. 

11.  Production   of  Disease. — Bacteria   which   produce 
diseases  are  known  as  pathogenic ;  those  which  do  not, 
as  non-pathogenic.     Between  the  two  groups  there  is  no 
sharp  line  of  separation,  for  true  pathogens  may  be  culti- 
vated under  such  adverse  conditions  that  their  virulence 
will  be  entirely  lost,  while  at  times  bacteria  ordinarily 
harmless  may  be  made  toxic  by  certain  manipulations  or 
by  introducing  them  into  animals  in  certain  combina- 
tions.    The  diseases  produced  are  the  result  of  the  sum 
of  numerous  activities  exhibited   by  the  bacteria.     For 
example,  it  may  be  that  a  microbe,  having  effected  its 


54  PATHOGENIC  BACTERIA. 

entrance  into  an  animal,  grows  with  great  rapidity, 
completely  blocking  up  the  blood-  and  lymph-channels, 
so  that  the  proper  circulation  of  these  fluids  is  stopped 
and  disease  and  death  must  result.  Perhaps  more  com- 
mon than  this  is  a  local  establishment  of  the  organisms, 
with  a  resulting  inflammation,  due  partly  to  the  presence 
of  the  foreign  organisms,  and  partly  to  their  toxic  me- 
tabolic products.  More  often,  however,  the  pathogenic 
bacteria  produce  powerful  metabolic  poisons — toxins, 
ptomaines,  etc. — which  either  cause  widespread  destruc- 
tion of  the  tissues  immediately  acted  upon,  or,  circulating 
throughout  the  organism,  produce  fever,  nervous  excita- 
tion, and  a  general  overthrow  of  the  normal  physiological 
equilibrium.  These  peculiarities  serve  to  divide  the  bac- 
teria into 

Septic  bacteria, 
Phlogistic  bacteria, 
Toxic  bacteria. 

The  bacteria  of  suppuration  probably  act  in  several 
ways.  Their  products  may  be  of  a  violently  chemotactic 
nature,  or  their  virulence,  exerted  upon  the  surrounding 
tissue,  may  destroy  large  numbers  of  the  cells,  whose 
dead  bodies  may  be  chemotactic.  When  the  suppura- 
tion is  violent  the  toxic  product  of  the  bacterium  is  itself 
most  probably  strongly  chemotactic. 

How  the  disease-producing  bacteria  effect  their  en- 
trance into  the  tissues  is  an  interesting  question.  The 
channels  naturally  open  to  them  are  those  leading  into 
the  interior  of  the  organism,  and  must  be  separately  con- 
sidered. 

(a)  The  Digestive  Tract. — Attention  has  already  been 
called  to  the  facility  with  which  the  bacteria  enter  the 
digestive  tract  in  foods  and  drinks.  Once  their  metabo- 
lism is  in  active  progress,  the  poisons  which  they  produce 
are  ready  for  absorption.  It  seems  probable  that  the 
absorption  of  the  toxic  substances  by  reducing  the  vitality 
of  the  individual  predisposes  to  the  formation  of  local 
lesions  through  which  the  bacteria  may  enter  the  intes- 


BIOLOGY  OF  BACTERIA.  55 

tinal  walls  to  continue  their  existence  and  produce 
greater  damage  than  before.  Some  such  theory  may 
explain  the  activity  of  such  organisms  as  those  of 
typhoid  and  cholera,  but  it  is  not  true  that  all  bacteria 
can  be  admitted  into  the  intestinal  structure  in  this  way. 
Before  reaching  the  intestine  the  bacteria  pass  through 
the  stomach,  and  must  resist  the  deleterious  action  of 
the  acid  gastric  juice,  which  few  are  able  to  do.  Eichhorst 
has  reported  an  epidemic  of  typhoid  fever  that  occurred 
in  a  military  barracks.  In  this  epidemic  the  infection 
seemed  to  take  place  through  the  rectum,  and  was  traced 
to  the  wearing  of  underclothing  previously  worn  by 
patients  and  improperly  washed. 

(6)  The  Respiratory  Tract. — Notwithstanding  the  moist 
interiors  of  the  mouth  and  nose  and  the  lashing  cilia  of 
the  pharyngeal  and  tracheal  mucous  membrane,  large 
numbers  of  bacteria  enter  the  smaller  bronchioles,  and 
sometimes  penetrate  as  deeply  as  the  air-cells.  It  is 
unusual  to  find  a  section  of  healthy  or  diseased  lung  in 
which  no  bacteria  can  be  found.  It  seems  to  have  been 
proven  by  Buchner  that  micro-organismal  infection  may 
take  place  through  the  lungs  without  definite  breach  of 
continuity  of  the  alveolar  walls.  He  mixed  anthrax 
spores  and  lycopodium  powder  together,  and  caused 
mice  and  guinea-pigs  to  inhale  them.  Out  of  the  66 
animals  used  in  his  experiments,  50  died  of  anthrax 
and  9  of  pneumonia.  Our  knowledge  of  the  disposition 
of  foreign  particles  in  the  lung  probably  explains  such  in- 
fection by  assuming  that  the  presence  of  the  lycopodium 
attracted  numerous  leucocytes  to  the  affected  air-cells, 
that  these  took  up  the  powder,  and  with  it  the  spores, 
and  that  the  leucocytes,  being  cells  of  very  susceptible 
animals,  were  unable  to  resist  the  growth  into  bacilli 
of  the  spores  which  they  had  carried  into  the  lymph- 
channels. 

On  the  other  hand,  it  has  been  shown  that  when 
the  entering  spores  are  unaccompanied  by  a  mechanical 
irritant  like  the  lycopodium  powder,  but  are  inspired 


56  PA  THOGENIC  BA  CTERIA. 

in  a  pulverized  liquid,  infection  takes  place  much  less 
readily. 

Tuberculosis  and  pneumonia  are  in  all  probability 
generally  the  result  of  the  inspiration  of  the  specific 
organisms. 

(c)  The  Skin  and  the  Superficial  Mucous  Membranes. — 
The  entrance  of  bacteria  into  the  tissues  by  way  of  the 
skin  is  probably  extremely  rare  if  the   skin   is   sound. 
Numerous  experimenters  have  caused  infection  by  rub- 
bing bacteria  or  their  spores  upon  the  skin.     It  would 
seem    probable   that   in   these    cases   there   must    have 
been  some  microscopic  lesions  into  which  the  bacteria 
were  forced.     My  own  investigations  have  shown  viru- 
lent staphylococci  of  suppuration  upon  the  conjunct! vse 
in  health.     It  is  very  improbable  that  the  bacteria  habit- 
ually present  upon  the  skin,  and  ready  to  enter  the  least 
abrasion,  can  penetrate  the  outer  coverings  of  the  body, 
except   when   disease   or   accident    has    rendered    them 
abnormally  thin  or  macerated. 

Turro  seems  to  have  shown  that  the  gonococcus  can 
enter  the  tissues  without  any  pre-existing  lesion,  for  he 
asserts  that  if  a  virulent  culture  simply  be  touched  to 
the  meatus  urinarius,  the  disease  will  be  established. 

(d)  Wounds. — The  results  of  the  entrance  of  bacteria 
into  unprotected  wounds  are  now  so   familiar  that  no 
one  deserving  of  the  name  of  surgeon  dares  to  allow  a 
wound  to  go  undressed. 

(e)  The  Placenta. — Very  frequently  the  occurrence  of 
specific   diseases   during   pregnancy  causes   abortion   of 
the  product  of  conception.     In  certain  cases  the  specific 
contagion  passes  through  the  placenta  and  infects  the 
fetus.     This  has   been   pretty  clearly  demonstrated   for 
variola,  malaria,  syphilis,  measles,  anthrax,  symptomatic 
anthrax,  glanders,  relapsing  fever,  typhoid,  and  in  rare 
cases  for  tuberculosis. 

Seeing  that  the  channels  by  which  bacteria  can  enter 
the  body  are  so  numerous,  and  that  there  is  scarce  a 
moment  when  some  part  of  us  is  not  in  contact  with 


BIOLOGY  OF  BACTERIA.  57 

them,  how  is  it  that  we  are  not  constantly  subject  to 
disease  ?  The  consideration  of  this  question,  together 
with  the  closely-related  questions  why  we  should  be 
subject  to  certain  diseases  only,  and  to  these  diseases 
at  certain  times  only,  must  be  reserved  for  another  chap- 
ter, where  the  subjects  Immunity  and  Susceptibility  can 
be  taken  up  at  length. 


CHAPTER   III. 

IMMUNITY  AND  SUSCEPTIBILITY. 

ONE  of  the  most  astonishing  facts  observed  in  physi- 
ology and  pathology  is  the  resistance  which  certain  ani- 
mals show  to  the  invasion  of  their  bodies  by  the  germs 
of  disease. 

Thus,  man  suffers  from  typhoid  fever,  cholera,  and 
other  infectious  diseases  which  are  never  observed  in  the 
domestic  animals;  cattle  are  subject  to  a  pleuro-pneumo- 
nia  which  does  not  affect  their  attendants;  man,  the  cow, 
and  the  guinea-pig  are  peculiarly  susceptible  to  tubercu- 
losis, which  the  cat,  dog,  and  horse  resist;  yellow  fever 
is  a  highly  contagious,  infectious  disease  which  is  almost 
certain  to  attack  all  new  arrivals  of  the  human  species 
when  epidemic,  but  which  rarely,  if  ever,  attacks  animals. 

The  popular  mind  accepts  the  statement  of  such  facts 
as  these  without  any  other  explanation  than  that  the 
animals  are  different,  and  so  of  course  their  diseases  are 
different;  but  the  more  the  scientific  man  contemplates 
them,  the  more  complicated  the  matter  becomes;  for, 
while  it  might  be  admitted  that  a  difference  in  the  body- 
temperature  and  chemistry  might  explain  why  a  frog 
will  resist  anthrax,  which  readily  kills  a  white  mouse,  it 
will  not  explain  why  a  house-mouse,  whose  chemistry 
must  be  almost  identical  with  that  of  the  white  mouse, 
can  successfully  combat  the  disease.  Nor  is  this  all. 
That  one  attack  of  yellow  fever,  of  typhoid  fever,  or 
of  scarlet  fever  renders  a  second  attack  almost  impos- 
sible is  not  the  less  interesting  because  of  its  every-day 
observation.  The  mouse  that  has  recovered  from  teta- 
nus will  not  take  tetanus  again,  and  most  interesting  and 

58 


IMMUNITY  AND  SUSCEPTIBILITY.  59 

extraordinary  is  the  fact  that  a  few  drops  of  blood  from 
the  recovered  mouse  injected  into  another  will  protect  it 
from  tetanus. 

Immunity  is  the  condition  in  which  the  body  of  an 
animal  resists  the  entrance  of  disease-producing  germs, 
or,  having  been  compelled  to  allow  them  to  enter,  resists 
their  growth  and  pathogenesis.  The  resistance  so  mani- 
fested is  a  distinct,  potential  vital  phenomenon. 

Susceptibility  is  the  opposite  condition,  in  which,  in- 
stead of  resistance,  there  is  a  passive  inertia  which  allows 
the  disease-producing  organisms  to  develop  without  oppo- 
sition. Susceptibility  is  accordingly  the  absence  of  im- 
munity. 

Immunity  is  either  natural  or  acquired. 

Natural  Immunity. — By  this  term  is  meant  the  natural 
and  constant  resistance  which  certain  healthy  animals 
exhibit  toward  certain  diseases. 

The  white  rat  is  peculiar  in  resisting  anthrax.  It  is 
almost  impossible  to  develop  anthrax  in  a  healthy  white 
rat,  but  Roger  found  that  such  an  animal  would  easily 
succumb  to  the  disease  if  compelled  to  turn  a  revolving 
wheel  until  exhausted.  Susceptibility  which  follows  such 
an  exhaustion  of  the  vital  powers  cannot  be  regarded  as 
other  than  accidental,  and  makes  no  exception  to  the 
statement  that  the  white  rat  is  immune  to  anthrax. 
Animals  such  as  man,  sheep,  cows,  rabbits,  and  white 
mice  are  susceptible  to  anthrax,  while  birds  and  reptiles 
are  generally  immune.  The  great  difference  in  the  morph- 
ology between  mammals  and  birds  and  reptiles,  together 
with  the  fact  that  their  temperature,  blood,  and  tissues 
all  differ,  makes  this  immunity  reasonably  intelligible. 
Morphological  differences,  however,  will  not  suffice  to 
explain  all  cases,  for  the  Caucasian  nearly  always  suc- 
cumbs to  yellow  fever,  while  the  negro  is  rarely  affected  ; 
and  scarlatina,  which  is  one  of  our  commonest  and  most 
dangerous  diseases  of  childhood,  is  said  to  be  unknown 
among  the  Japanese.  Nor  is  this  all,  for,  close  as  is  their 
resemblance  in  all  respects  except  color,  the  house-mouse, 


60  PA  THOGENIC  BA  CTERIA. 

field-mouse,  and  white  mouse  differ  very  much  in  their 
susceptibility  to  various  diseases. 

Acquired  immunity  is  resistance  which  is  the  result 
of  accidental  circumstances.     It  may  result — 

A.  By  recovery  from  a  mild   attack  of  the   disease. 
Most  adults  have  suffered  from  rubeola,  scarlatina,  and 
varicella  in  childhood,  and  in  consequence  of  the  attacks 
are  now  immune  to  these  diseases — i.  e.  will  not  become 
affected  again.     One  attack  of  yellow  fever  is  always  a 
complete  guard  against  another.     Typhoid  fever  is  rarely 
followed  by  a  second  attack. 

B.  By  recovery  from  an  attack  of  a  slightly  different 
disease.     Sometimes  the  immunity  is  experimentally  pro- 
duced, as  when  by  vaccination  we  produce  the  vaccine 
disease  and  afterward  resist  variola.    Acquired  immunity 
is  a  little  less  complete  and  not  so  permanent  as  natural 
immunity,  for  in  the  latter  it  is  only  when  the  functions 
of  the  individual  are  disturbed  or  his  vitality  depressed 
that  the  resistance  is  lost,  while  in  the  former  time  seems 
to  lessen  the  power  of  resistance,   so  that  rubeola  and 
scarlatina  may  return  in  a  few  months  or  years,  and  for 
complete  protection  vaccination  may  need  to  be  done  as 
often  as  every  seven  years. 

C.  By    the    injection   of    antitoxic    substances.      At 
present    there   is   much    agitation    over   the    newly-dis- 
covered antitoxin  of  diphtheria,  the  injection  of  about 
2  c.cm.  of  which  will  give  complete  protection  against 
the   disease   for  a  period   lasting   from  a  month  to  six 
weeks. 

Immunity  may  be  destroyed  in  numerous  ways: 
(a)  By  variation  from  the  normal  temperature  of  the 
animal  under  observation.  Pasteur  observed  that  chick- 
ens would  not  take  anthrax,  and  suspected  that  this 
immunity  might  be  due  to  their  high  body-temperature. 
After  inoculation  he  plunged  the  birds  into  a  cold  bath, 
reduced  their  temperature,  and  succeeded  in  destroying 
their  immunity.  The  experiment  was  a  success,  but  the 
reasoning  seems  to  have  been  faulty,  as  the  sparrow, 


IMMUNITY  AND  SUSCEPTIBILITY.  6 1 

with  a  temperature  equally  high,  readily  falls  a  victim 
to  anthrax  without  a  cold  bath. 

(b}  By  altering  the  chemistry  of  the  blood  by  changing 
the  diet  or  by  hypodermic  injection.  L,eo  found  that 
when  white  rats  were  injected  with  or  fed  upon  phlorid- 
zin  an  artificial  glycosuria  resulted  which  destroyed  their 
natural  resistance  to  anthrax.  Hankin  found  that  rats, 
which  possess  considerable  immunity  to  anthrax,  could 
be  made  susceptible  by  a  diet  of  bread.  Platania  suc- 
ceeded in  producing  anthrax  in  dogs,  frogs,  and  pigeons, 
naturally  immune,  by  subjecting  them  to  the  influence 
of  curare,  chloral,  and  alcohol. 

(c)  By  diminishing  the  strength  of  the  animal.  Roger 
by  compelling  white  rats  to  turn  a  revolving  wheel  until 
exhausted  destroyed  their  immunity  to  anthrax. 

(ct)  By  removing  the  spleen.  Bardach  has  shown  that 
the  chances  of  recovery  from  specific  diseases  are  greatly 
lessened  by  the  removal  of  the  spleen. 

(e)  By  combining  two  different  species  of  bacteria,  either 
of  which,  when  injected  alone,  would  be  harmless  or  of 
slight  effect.  Roger  found  that  when  animals  immune 
to  malignant  edema  were  simultaneously  injected  with 
i  to  2  c.  cm.  of  a  culture  of  Bacillus  prodigiosus  and  the 
bacillus  of  malignant  edema,  they  would  contract  the 
disease.  Pawlowski  found  that  when  rabbits,  which 
are  very  susceptible  to  anthrax,  were  simultaneously  in- 
jected with  anthrax  and  prodigiosus,  they  recovered 
from  the  anthrax,  as  if  the  harmless  microbe  possessed 
the  power  of  neutralizing  the  products  of  the  patho- 
genic form. 

Sometimes  an  apparent  immunity  depends  upon  the 
attenuation  of  the  culture  used  for  inoculation,  and  the 
erroneous  results  to  which  such  a  mistake  may  lead  are 
obvious.  Should  a  culture  become  attenuated,  its  viru- 
lence may  sometimes  be  increased  by  inoculating  it  into 
the  most  susceptible  animal,  then  from  this  to  a  less 
susceptible,  and  then  to  an  immune  animal.  The  viru- 
lence of  anthrax  is  increased  by  inoculation  into  pigeons, 


62  PATHOGENIC  BACTERIA. 

and  also  by  cultivation  in  an  infusion  of  the  tissues  of 
an  animal  similar  to  the  one  to  be  inoculated. 

It  must  be  understood  that  the  term  "immunity"  is 
a  relative  one,  and  that  while  ua  white  rat  is  immune 
against  anthrax  in  amounts  sufficiently  large  to  kill  a 
rabbit,  it  is  perhaps  not  immune  against  a  quantity 
sufficiently  large  to  kill  an  elephant." 

It  is  not  to  be  expected  that  such  intricate  phenomena 
as  these  which  have  been  mentioned  could  be  observed 
and  suffered  to  go  unexplained.  We  have  explanations, 
but,  unfortunately,  they  are  as  intricate  as  the  phenomena, 
and,  though  each  may  possess  its  grain  of  truth,  not  one 
will  satisfy  the  demands  of  the  thoughtful  student.  In 
brief  review,  the  theories  of  immunity  are  the  following : 

1.  THE  EXHAUSTION  THEORY. — This  hypothesis  was 
advanced  by  Pasteur  in  1880,  and  suggests  that  by  its 
growth  in  the  body  the  micro-organism   uses  up  some 
substance  essential  to  its  life,  and  that  when  this  sub- 
stance is  exhausted  the  microbe  can  no  longer  thrive. 
The  removal  of  the  necessary  material,  if  complete,  will 
cause  permanent  immunity. 

As  Sternberg  points  out,  were  this  theory  true  we  must 
have  within  us  a  material  of  small-pox,  a  material  of 
measles,  a  material  of  scarlet  fever,  etc.,  to  be  exhausted 
by  its  appropriate  organism.  It  would  necessitate  an 
almost  inconceivably  complex  body-chemistry  and  a 
rather  stable  condition  of  the  same. 

2.  THE   RETENTION    THEORY. — In    the    same    year 
Chauveau   suggested   that   the   growth   of    the   bacteria 
in  the  body  might  originate  some  substance  prejudicial 
to  their  further  and  future  development.     There  seems 
to  be  a  large  kernel  of  truth  in  this,  but  were  it  always 
the  case  we  would  have  added  to  our  blood  a  material 
of  small-pox,  a  material  of  measles,  a  material  of  scarlet 
fever,  etc. ,  so  that  we  would  become  saturated  with  the 
excrementitious  products  of  the  bacteria,  instead  of  hav- 
ing so  many  substances  subtracted  from  our  chemistry. 

3.  THE  THEORY  OF  PHAGOCYTOSIS. — In   1881,  Carl 


IMMUNITY  AND  SUSCEPTIBILITY.  63 

Roser  suggested  a  relation  between  immunity  and  the 
already  familiar  phenomenon  of  phagocytosis.  Stern- 
berg  in  the  United  States  and  Koch  in  Germany  observed 
the  same  thing,  but  little  real  attention  was  paid  to  the 
subject  until  1884,  when  Metchnikoff  appeared,  with  his 
careful  observations  upon  the  daphnia,  as  the  great  cham- 
pion of  the  theory  which  is  now  known  as  * (  Metchni- 
koff's  theory  of  phagocytosis." 

Phagocytosis  is  the  swallowing  or  incorporating  of 
particles  by  certain  of  the  body-cells  which  are  called 
phagocytes.  This  activity  of  the  cells  toward  inert 
particles  had  been  observed  by  Virchow  as  early  as  1840, 
and  toward  living  bacteria  by  Koch  in  1878,  but  was  not 
carefully  studied  until  1884.  Metchnikoff  divides  the 
phagocytes  into  fixed  phagocytes,  comprising  the  fixed 
connective-tissue  cells,  endothelium,  etc.,  and  the  free 
phagocytes,  which  are  the  leucocytes.  The  terms  "phag- 
ocyte" and  "leucocyte"  are  not  to  be  regarded  as  synon- 
ymous in  this  connection  ;  all  leucocytes  are  not  phag- 
ocytic,  the  lymphocyte  having  never  been  observed  to 
take  up  bacteria. 

It  is  obvious  that  only  those  cells  can  be  phagocytic 
which  are  without  a  resisting  cell-wall  and  possess 
ameboid  movement.  When  an  ameba,  in  a  liquid  con- 
taining numerous  diatoms  and  bacteria,  is  watched 
through  the  microscope,  an  interesting  phenomenon  is 
observed.  The  ameba  will  approach  one  of  the  vege- 
table cells,  even  though  it  may  be  at  a  distance,  will 
apprehend  and  surround  it,  and  within  the  animal  cell 
the  vegetable  cell  will  be  digested  and  assimilated.  The 
ameba  has  no  eyes,  no  nose,  no  volition,  and,  so  far  as 
we  can  determine,  no  nervous  apparatus  which  gives 
it  tactile  sense,  yet  it  will  approach  the  particle  fitted 
for  its  use  and  swallow  it.  The  attraction  which  draws 
the  cells  together  has  been  called  by  Peffer  chemotaxis, 
chemiotaxis,  or  chemotropism. 

Chemotaxis  is  the  exhibition  of  an  attractive  force 
between  cells  and  their  nutriment,  ameboid  cells  and 


64  PATHOGENIC  BACTERIA. 

food-particles,  and  sometimes  between  ameboid  cells  and 
inert  particles.  This  attractive  force,  when  operating  so 
as  to  draw  the  ameba  to  the  particle  it  will  devour,  is 
further  named  positive  chemotaxis  in  order  to  distinguish 
it  from  a  repulsive  force  sometimes  exerted  causing  the 
ameboid  cells  to  fly  from  an  enemy,  as  it  were,  and  which 
is  called  negative  chemotaxis. 

The  force  that  operates  and  guides  the  ameba  in  its 
movements  is  exactly  the  same  as  that  which  governs  the 
movement  of  the  phagocytic  cells  of  the  human  body, 
and  observation  of  these  phenomena  is  not  difficult.  If 
a  small  capillary  tube  be  filled  with  sweet  oil  and  placed 
beneath  the  skin,  only  a  short  time  need  pass  before  it 
will  be  found  full  of  leucocytes — positive  chemotaxis. 
If,  instead  of  sweet  oil,  oil  of  turpentine  be  used,  not 
a  leucocyte  will  be  found — negative  chemotaxis. 

Phagocytosis  is  almost  universal  in  the  micro-or- 
ganismal  diseases  at  some  stage  or  another.  If  the 
blood  of  a  patient  suffering  from  relapsing  fever  be 
studied  beneath  the  microscope,  it  will  be  found  to 
contain  numerous  active  mobile  spirilla,  all  free  in  the 
liquid  portion  of  the  blood.  As  soon  as  the  apyretic 
stage  comes  on  not  a  single  free  spirillum  can  be  found. 
Every  one  is  seen  to  be  enclosed  in  the  leucocytes. 

At  the  edge  of  an  erysipelatous  patch  a  most  active 
warfare  is  waged  between  the  streptococci  and  the  cells. 
Near  the  centre  of  the  patch  there  are  many  free  strep- 
tococci and  a  few  cells.  At  the  margin  there  are  free 
streptococci,  and  also  a  great  many  streptococci  en- 
closed in  cells  (leucocytes)  which  are,  for  the  most  part, 
dead.  In  the  newly-invaded  tissue  we  find  hosts  of 
active  living  cells  engaged  in  eating  up  the  enemies 
as  fast  as  they  can.  The  phagocytologists  tell  us  that  at 
the  centre  the  bacteria  are  fortified,  actively  growing,  and 
virulent  ;  in  the  next  zone  the  leucocytes  which  have 
feasted  upon  the  bacteria  are  poisoned  by  them  ;  outside, 
the  cells,  which  are  more  powerful  and  which  are  con- 
stantly being  reinforced,  are  waging  successful  warfare 


IMMUNITY  AND  SUSCEPTIBILITY.  65 

against  the  streptococci.  In  this  manner  the  battle  con- 
tinues, the  cells  now  being  obliged  to  yield  to  the  bacteria 
and  the  patch  spreading,  while  the  cells  subsequently  re- 
inforce and  destroy  the  bacteria,  so  that  the  disease  comes 
to  a  termination. 

Metchnikoff  introduced  fragments  of  tissue  from  ani- 
mals dead  of  anthrax  under  the  skin  of  the  back  of  a  frog, 
and  found  it  surrounded  and  penetrated  by  leucocytes  con- 
taining many  of  the  bacilli. 

It  need  scarcely  be  pointed  out  that  a  loophole  of  doubt 
exists  in  all  these  illustrations:  the  bacteria  may  have  been 
dead  before  the  cells  ingested  them,  and  the  phenomena  of 
digestion  and  destruction  which  have  gone  on  in  their  in- 
teriors may  have  been  exerted  upon  dead  bacteria.  To  the 
relapsing- fever  illustration  we  may  take  exceptions,  and 
state  that  the  apyrexia  may  have  marked  the  death  of 
the  spirilla,  which  were  taken  up  by  the  leucocytes  only 
when  dead.  In  the  erysipelas  illustration  the  streptococci 
remote  from  the  centre  of  the  lesion  may  have  met  from 
the  body-juices  or  some  other  cause  a  more  speedy  death 
than  that  from  the  digestive  juices  of  the  leucocyte. 

Metchnikoff,  however,  is  prepared  to  show  us  that  the 
leucocytes  do  take  up  living  pathogenic  organisms.  He 
succeeded  in  isolating  two  leucocytes,  each  containing  an 
anthrax  spore,  and  conveying  them  to  artificial  culture- 
media,  where  he  watched  them.  The  new  environment 
being  better  adapted  to  the  growth  of  the  spore  than  for 
the  nourishment  of  the  leucocyte,  the  latter  died,  and 
the  spore  developed  under  his  eyes  into  a  healthy  bacillus. 
Seeing  that  the  animal  cells  take  up  bacteria,  and  seeing 
that  the  ameba  can  ingest  and  digest  "threads  of  lepto- 
thrix  ten  times  as  long  as  itself,"  we  need  only  put  two 
and  two  together  to  see  that  Metchnikoff 's  theory  rests 
upon  a  very  substantial  foundation.  The  more  virulent 
the  bacteria,  the  less  ready  the  leucocytes  are  to  seize 
them.  The  more  immune  the  animal,  the  greater  is  the 
affinity  of  the  leucocyte  for  the  bacteria. 

The  organisms  which  are  seized  upon  by  the  leucocytes 


66  PATHOGENIC  BACTERIA. 

do  not  remain  in  the  blood,  but  are  collected  in  the  spleen 
and  the  lymphatic  glands;  and  not  the  least  important 
fact  in  favor  of  phagocytosis  is  that  observed  by  Bardach, 
that  excision  of  the  spleen  diminishes  the  resistance  to 
infectious  disease. 

Quinin  also  furnishes  a  therapeutic  support  to  the 
theory.  It  is  known  that  quinin  increases  the  destruc- 
tion of  leucocytes.  Woodhead  inoculated  a  number  of 
rabbits  with  anthrax,  giving  quinin  to  some  of  them. 
Those  which  had  received  the  drug  died  earliest — a 
result  probably  dependent  upon  the  destruction  of  part 
of  the  phagocytic  army. 

Ruffer  found  that  the  "phagocytes  evince  a  distinct 
selective  tendency  between  various  kinds  of  organisms. 
They  will  leave  the  bacillus  of  tetanus  in  order  to  seize 
upon  the  Bacillus  prodigiosus  if  simultaneously  intro- 
duced; also  the  streptococci  in  diphtheria  for  the  Klebs- 
Loffler  bacilli.  This  is  illustrated  in  the  diphtheritic 
membrane,  where  at  the  surface  one  can  see  leucocytes 
taking  in  numbers  of  the  bacilli,  but  leaving  the  strepto- 
cocci almost  untouched,  with  the  immediate  result  that 
streptococci  are  often  found  in  the  deeper  parts  of  the 
membrane,  and  with  the  remote  result  that  secondary 
abscesses  occurring  in  the  course  of  diphtheria  are  never 
due  to  the  bacillus  of  diphtheria,  but  to  some  other  or- 
ganism. ' ' 

Hankin  and  Hardy  found  that  the  three  varieties  of 
leucocytes  in  the  frog's  blood  play  important  parts  in  the 
destruction  of  anthrax  bacilli,  this  destructive  process 
being  accomplished  thus : 

1.  The  eosinophile  cells  are  first  to  approach  and  swal- 
low  the  bacteria.      As  this  takes  place  the  eosinophile 
granules  are  seen  to  dissolve  and  act  upon  the  bacteria. 

2.  The  hyaline  cells  take  up  the  remains  of  the  bac- 
teria destroyed  by  the  eosinophile  leucocytes. 

3.  The  basophile  cells  come  to  the  field  loaded  with 
basophilic    granules,    supposed    to   be   antidotal    to   the 
poisons  of  the  bacteria,  surround  the  combatants,  neu- 


IMMUNITY  AND  SUSCEPTIBILITY.  67 

tralize  the  bacterial  poisons,  and  liberate  the  contesting 
cells. 

Wyssokowitsch  found  that  saprophytic  micro-organ* 
isms  are  quickly  eliminated  from  the  blood  when  in- 
jected into  the  circulation.  This  elimination  is  not 
by  excretion  through  organs  nor  by  destruction  in  the 
streaming  blood,  but  by  collection  in  the  small  capil- 
laries, where  the  blood-stream  is  slow  and  where  the 
micro-organisms  are  taken  up  by  the  endothelial  cells. 
Wyssokowitsch  found  them  most  numerous  in  the  liver, 
spleen,  and  bone-marrow,  and  found  that  in  these  situa- 
tions they  were  destroyed  in  a  short  time — saprophytic 
in  a  few  hours,  pathogenic  in  from  twenty-four  to  forty- 
eight  hours.  Spores  of  Bacillus  subtilis  remained  as 
living  entities  in  the  spleen  for  three  months. 

4.  THE  HUMORAL  THEORY. — It  was  observed  that  if 
anthrax  bacilli  were  introduced  into  a  few  drops  of 
rabbit's  blood,  they  were  instantly  killed.  This  obser- 
vation was  one  of  immense  importance,  and  from  it  and 
similar  observations  Buchner  deduced  the  principles  of 
his  theory,  which  teaches  that  the  destruction  of  patho- 
genic bacteria  in  the  body  is  due  to  the  bactericidal 
action  of  the  blood-plasma,  not  to  phagocytosis;  which 
phenomenon  amounts  to  nothing  more  than  the  burial 
of  the  dead  bacteria  in  "cellular  charnel-houses."  The 
experiments  of  Buchner  and  his  followers  have  shown 
that  freshly-drawn  blood,  blood-plasma,  defibrinated 
blood,  aqueous  humor,  tears,  milk,  urine,  and  saliva 
possess  marked  destructive  influence  upon  the  organ- 
isms brought  in  contact  with  them — an  influence  easily 
destroyed  by  heat. 

The  apparent  paradox  of  rapid  multiplication  of  an- 
thrax bacilli  in  the  rabbit's  blood  enclosed  in  the  rabbit's 
body,  and  the  reversed  action  in  the  test-tube,  caused  im- 
mediate and  prolonged  opposition  to  the  theory.  Each  side 
of  the  question  seemed  well  supported.  The  phagocytolo- 
gists,  however,  showed  that  bacteria  were  often  injured 
and  their  vegetative  powers  destroyed  by  sudden  changes 


68  PATHOGENIC  BACTERIA. 

from  one  culture-medium  to  another,  this  being  proved 
by  Haff  kine,  who  in  experimenting  with  aqueous  humor 
has  shown  that  its  germicidal  actions  are  largely  imagin- 
ary, and  due  to  the  dispersion  of  the  organisms  in  a  large 
amount  of  watery  liquid.  When  the  micro-organisms 
are  introduced  into  it  in  such  a  manner  as  to  remain 
together,  they  grow  well.  If  the  tube  be  shaken,  so  as 
to  distribute  them,  they  die.  Again,  Adami  has  shown 
that  when  blood  is  shed  there  is  almost  always  a  pro- 
nounced destruction  of  corpuscles,  and  suggests  that  the 
antibiotic  property  of  the  shed  blood  may  be  due  to 
solution  of  the  nucleins  formerly  in  the  substance  of  the 
leucocytes.  Jetter  endeavored  to  prove  the  germicidal 
action  of  the  serum  to  be  due  to  certain  salts  which  it 
contained.  His  experiments,  which  consisted  in  observ- 
ing the  action  of  solutions  of  various  salts  in  mixtures 
of  water,  glycerin,  and  gelatin,  were  justly  condemned 
by  Buchner  on  the  ground  that  such  mixtures,  though 
they  might  contain  constituents  of  blood-serum,  were  far 
from  approximating  the  normal  serum  in  composition. 

Wyssokowitsch,  however,  surely  argued  against  hu- 
moral germicide  when  he  showed  that  the  spores  of  Ba- 
cillus subtilis  could  reside  in  the  spleen  for  three  months 
uninjured. 

In  supporting  their  theory  the  humoralists  experimented 
by  placing  beneath  the  skin  micro-organisms  enclosed  in 
little  bags  of  pith,  collodium,  etc.  These  bags  allowed 
the  fluids  of  the  body  free  access  to  the  bacteria,  but 
would  shut  out  the  phagocytes.  By  these  means  Hiippe 
and  Iviibarsch  have  repeatedly  seen  the  bacteria  grow 
well,  while  the  attempts  of  Baumgarten  have  failed. 
Such  experiments  are  by  no  means  conclusive,  for  we 
should  remember  that  the  operation  necessary  and  the 
presence  of  the  foreign  body  in  which  the  bacteria  are 
encased  produce  an  inflammatory  transudate  which  may 
have  properties  very  different*  from  those  of  the  normal 
juices. 

How  much  of  the  immunity  which  animals  enjoy  de- 


IMMUNITY  AND  SUSCEPTIBILITY.  69 

pends  upon  the  antibactericidal  action  of  their  body- 
juices  must  remain  an  open  question.  In  some  cases  the 
germicidal  action  of  the  blood  seems  to  be  unquestion- 
able. Buchner  has  shown  that  the  blood-serum  of  ani- 
mals only  possesses  this  germicidal  power  when  freshly 
drawn,  and  that  exposure  of  the  serum  to  sunlight,  its 
mixture  with  the  serum  from  another  species  of  animal, 
its  mixture  with  distilled  water  or  with  dissolved  cor- 
puscles, and  heating  it  to  55°  C.,  check  the  bactericidal 
power.  Buchner  also  points  out  that  the  bactericidal 
and  globulicidal  actions  of  the  blood  are  simultaneously 
extinguished. 

Much  discussion  has  arisen  as  to  exactly  what  the  pro- 
tective substances  are.  Buchner  has  applied  the  term 
alexin  to  the  protective  proteid  substances  found  in  the 
blood  of  naturally  immune  animals.  Hankin  has  given 
us,  together  with  an  extension  of  Buchner' s  idea,  a  con- 
siderable nomenclature  of  somewhat  questionable  utility. 
He  divides  the  protective  substances  (alexins)  into  sozins, 
which  occur  in  the  blood  of  animals  with  natural  immu- 
nity, and  phylaxins,  which  occur  in  the  blood  of  animals 
with  acquired  immunity.  Both  sozins  and  phylaxins  are 
divisible  into  two  groups — thus :  a  sozin  which  acts  de- 
structively upon  bacteria  is  called  a  myco-sozin;  one 
which  neutralizes  bacterial  poisons,  a  toxo-sozin.  A  phy- 
laxin  which  acts  destructively  upon  bacteria  is  called  a 
myco-phylaxin ;  one  which  neutralizes  bacterial  toxins, 
a  toxo-phylaxin.  A  glance  will  show  that  this  classifi- 
cation is  based  upon  the  somewhat  doubtful  existence 
of  alexins. 

5.  THE  THEORY  OF  ANTITOXINS. — It  is  a  well-known 
fact  that  individuals  can  accustom  themselves  to  the  use 
of  certain  poisons,  as  tobacco,  opium,  and  arsenic,  so  as 
to  experience  no  inconvenience  from  what  would  be  poi- 
sonous doses  for  other  individuals.  This  is  purely  a  matter 
of  tolerance,  but  is  of  interest  in  connection  with  the 
observations  which  are  to  follow. 

Ehrlich  has  shown  that  animals  can  tolerate  gradually- 


70  PATHOGENIC  BACTERIA, 

increasing  doses  of  ricin  and  abrin,  provided  that  up  to 
a  certain  point  the  increase  of  dosage  is  very  small. 
When  this  point  is,  however,  safely  passed,  the  increase 
in  dosage  can  be  very  rapid,  yet  without  signs  of  poison- 
ing, seemingly  because  the  drug  is  no  longer  simply  tol- 
erated, but  tolerated  and  simultaneously  neutralized.  By 
experimentation  Khrlich  has  shown  that  during  the 
period  of  simple  tolerance  the  blood  of  the  animal  is 
unaltered,  but  that  as  soon  as  the  tolerance  becomes 
unlimited  the  blood  contains  a  new  substance,  capable 
not  only  of  protecting  the  animal  by  which  it  is  pro- 
duced, but  also  other  animals  into  whose  blood  it  is  in- 
troduced. In  the  ricin  experiments  this  substance  was 
described  as  antiricin  ;  in  the  experiments  with  abrin,  as 
antiabrin. 

These  investigations  of  Ehrlich  with  the  poisons  of 
higher  plants  succeeded,  but  threw  much  light  upon,  the 
extraordinary  work  of  Behring,  Wernicke,  and  Kitasato, 
who  experimented  with  the  toxins  of  diphtheria  and 
tetanus,  and  showed  that  in  the  blood  of  animals  accus- 
tomed to  these  poisons,  new  substances — antitoxins,  found 
by  Brieger  to  be  proteid  in  nature — were  produced. 

The  antitoxic  theory  of  immunity,  being,  in  the  cases 
cited  at  least,  a  fact  capable  of  demonstration,  has  estab- 
lished itself  at  present  as  the  most  important  hypothesis. 
According  to  it,  acquired  immunity,  at  least,  depends  upon 
the  development  in  the  blood  of  a  neutralizing  substance 
probably  related  to  the  nucleins. 

It  is  of  prime  importance  to  remember  that  the  anti- 
toxin is  an  entirely  new  substance  which  does  not  occur 
in  the  blood  of  normal  animals. 

The  difference  between  this  theory  of  neutralization 
by  antitoxins  and  Chaveau's  retention  hypothesis  is  quite 
marked.  The  retention  theory  teaches  that  a  bacterium 
leaves  behind  it  a  substance  prejudicial  to  its  future 
growth  in  the  economy — a  distinct  metabolic  product. 
The  antitoxic  theory  shows  the  protective  substance  to 
be  a  product  not  of  bacterial  growth,  but  of  tissue-energy, 


IMMUNITY  AND  SUSCEPTIBILITY.  71 

not  depending  upon  the  presence  of  the  bacteria,  but 
upon  the  presence  of  a  poison. 

The  antitoxins  do  not  act  harmfully  upon  the  bacteria, 
do  not  preclude  their  growth  in  the  animal  body,  but 
prevent  their  pathogenesis  by  annulling  their  toxicity — 
i.  e.  enabling  the  body-cells  to  endure  the  injury — and 
placing  them  in  a  position  exactly  parallel  with  non- 
pathogenic  bacteria. 

The  diseases  which  are  at  present  controllable  by  anti- 
toxins are  toxic  diseases,  caused  by  the  entrance  of  toxin- 
producing  bacteria  into  the  body.  The  growth  of  these 
toxin-producers  probably  depends  upon  the  inability  of 
the  body-cells  or  bactericidal  body-juices  to  properly  cope 
with  them,  so  that  they  develop  and  engender  the  poison- 
ous substances  which  are  the  essential  factors  of  disease- 
production.  The  more  the  body  and  its  component  ele- 
ments are  injured,  the  more  successful  the  inroads  of  the 
bacteria,  the  more  prolific  the  toxin-production,  and  the 
more  severe  the  affection. 

The  presence  of  the  antitoxin  annuls  the  poison,  main- 
tains the  vitality  of  the  organism  as  a  whole,  sustains 
the  integrity  of  its  tissues,  and  so  places  the  pathogenic 
bacterium  on  a  very  different  footing  in  relation  to  the 
organism. 

An  antitoxin  is  a  neutralizing  or  annulling  agent,  not 
a  regenerating  one,  and  therefore  in  therapeutics  finds 
its  proper  sphere  only  in  the  beginning  of  the  disease 
combated.  Up  to  a  certain  point  the  symptoms  of  diph- 
theria and  tetanus  are  due  to  the  circulation  of  toxins  in 
the  blood,  and  can  be  successfully  combated  by  antitoxic 
neutralization.  Later  in  both  diseases  we  have  symp- 
toms resulting  from  disorganization  of  the  nervous  sys- 
tem, degeneration  of  the  heart-muscle,  destruction  of  the 
kidneys,  etc. ,  and  the  neutralization  of  the  poison  can  be 
of  no  avail  because  the  lesions  are  irreparable,  and  the 
patient  must  succumb. 

I  have  used  the  term  "neutralization,"  in  speaking  of 
the  antitoxins,  in  a  rather  free  and  scarcely  warranted 


72  PA  THOGENIC  BA CTERIA. 

manner,  and  must  call  attention  to  the  fact  that  their 
operation  is  in  no  way  analogous  to  chemical  neutraliza- 
tion. From  mixtures  of  toxin  and  antitoxin  the  un- 
changed poison  has  been  recovered.  The  effect  of  an 
antitoxin,  unlike  that  of  a  toxo-phylaxin,  seems  to  be  a 
biologic  one,  by  which  the  tissues  are  so  stimulated  as  to 
endure  the  action  of  a  substance  ordinarily  disorganizing 
in  effect. 

Buchner  and  Roux  have  both  pointed  out  that  when 
the  toxins  and  antitoxins  are  mixed  and  introduced  into 
animals  of  greater  susceptibility  than  are  ordinarily  used, 
the  presence  of  an  unaltered  toxin  can  easily  be  demon- 
strated. 

According  to  Buchner,  the  antitoxins  differ  from  the 
alexins  in  being  new  substances  in  the  blood,  in  being 
without  germicidal  or  chemical  neutralizing  power  against 
the  toxins,  and  in  being  stable  compounds  which  can 
resist  heat  to  75°  C.,  can  resist  a  reasonable  amount  of 
exposure  to  light,  and  which  are  not  altered  by  decompo- 
sition of  the  substances  containing  them. 

The  antitoxins  are  specific  for  one  poison  only.  Ehrlich 
found  that  antiricin  was  powerless  against  abrin,  and  vice 
versd.  Diphtheria  antitoxin  is  of  no  avail  against  tetanus, 
and  vice  versd. 

The  immunity  which  the  antitoxins  produce  is  fuga- 
cious, varying  considerably  according  to  the  particular 
substance  employed.  As  a  rule,  it  is  limited  to  a  few 
months — at  least  in  the  case  of  such  antitoxins  as  we  can 
produce  experimentally. 

From  all  that  has  gone  before  it  must  be  clear  to  the 
reader  that  no  single  theory  thus  far  advanced  can  ex- 
plain immunity.  Acquired  immunity  may  depend  in 
the  great  majority  of  cases  upon  antitoxins,  but  as  yet 
we  have  no  satisfactory  explanation  of  natural  immunity. 
The  humoral  theory  may  be  applicable  in  some  cases  ;  in 
others  one  cannot  deny  the  importance  of  the  role  played 
by  the  phagocytes. 


CHAPTER    IV. 
METHODS   OF  OBSERVING  BACTERIA. 

WHOEVER  would  study  bacteria  must  be  equipped  with 
a  good  microscope.  The  instruments  generally  provided 
for  the  use  of  medical  students  in  college  laboratories,  as 
well  as  those  seldom-employed  u  show  microscopes  "  seen 
in  physicians'  offices,  are  ill  adapted  for  the  purpose. 
The  essential  features  of  a  bacteriological  instrument 
are  lenses  giving  a  clear  magnification  extending  as 
high  as  one  thousand  diameters,  and  a  good  condenser 
for  intensifying  the  lights  thrown  upon  the  objects.  It 
naturally  follows  that  the  best  work  requires  the  best 
lenses.  The  cheapest  good  microscope  which  is  at  pres- 
ent offered  to  the  public  is  the  BB.  Continental  stand, 
made  by  Bausch  and  L,omb.  This  stand  is  provided  with 
everything  necessary,  is  fitted  with  very  creditable  objec- 
tives, including  an  excellent  Ty  oil-immersion  lens,  and 
seems  capable  of  doing  very  good  work.  I  do  not 
recommend  this  as  the  best  instrument  obtainable,  but 
as  one  that  is  both  good  and  cheap.  For  those  who  desire 
the  very  best  the  rather  costly  outfits  made  by  Carl  Zeiss 
of  Jena  are  unexcelled. 

For  those  who  may  begin  the  use  of  the  Abbe  con- 
denser and  oil-immersion  lenses  without  the  advantage 
of  personal  instruction  a  few  hints  will  not  be  out  of 
place : 

Always  employ  good  slides  without  bubbles,  and  thin 
cover-glasses ;  No.  i  are  best. 

Place  a  drop  of  oil  of  cedar  upon  the  cover-glass  of 
the  specimen  to  be  examined  ;  rack  the  body  of  the  instru- 
ment down  until  the  oil-immersion  lens  touches  the  oil ; 

73 


74  PA  THOGENIC  BA  CTERIA . 

keep  on  until  it  almost  touches  the  glass,  then  look  into 
the  microscope  and  find  the  object  by  slowly  and  firmly 
racking  up.  As  soon  as  the  object  comes  into  view 
leave  the  rack  and  pinion  and  focus  with  the  fine  adjust- 
ment. 

Always  select  the  light  from  a  white  cloud  if  possible  ; 
if  there  are  no  white  clouds,  choose  the  clearest  whitest 
light  possible.  Never  under  any  circumstances  employ 
sunlight,  which  is  ruinous  to  the  eyes  and  useful  only 
for  photomicrography. 

In  using  low-power  lenses  the  Abbe  condenser  must  be 
moved  away  from  the  object  and  the  light  modified  by 
the  iris-diaphragm.  The  distance  between  condenser  and 
object  should  correspond  more  or  less  closely  with  the 
distance  between  objective  and  object. 

In  using  high  powers  the  Abbe  condenser  must  be 
brought  near  the  object  and  the  light  modified  by  the 
iris-diaphragm. 

If  the  oil-immersion  lens  is  used,  it  is  perhaps  best  to 
employ  the  plane  side  of  the  mirror.  When  with  this 
lens  a  section  of  tissue  is  examined  for  details,  the  light 
must  be  modified  by  the  iris-diaphragm,  opening  and 
closing  it  alternately  until  the  best  effect  of  illumina- 
tion is  achieved.  If  tissue  be  searched  for  stained  bac- 
teria, and  no  cellular  detail  is 'required,  the  diaphragm 
should  be  wide  open  to  admit  a  great  flood  of  light 
and  extinguish  everything  except  the  deeply-colored 
bacteria. 

When  unstained  bacteria  are  to  be  examined  with  the 
oil-immersion  lens,  the  diaphragm  should  be  closed  so 
as  to  leave  only  a  small  opening  through  which  the 
light  can  pass. 

Bacteria  may  be  examined  either  stained  or  unstained. 
The  former  condition  would  always  be  preferable  if  the 
process  of  coloring  the  organisms  did  not  injure  them. 
Unfortunately,  it  is  generally  the  case  that  the  drying, 
heating,  boiling,  macerating,  and  acidulating  to  which 
we  expose  the  organisms  in  the  process  of  staining  alter 


METHODS  OF  OBSERVING  BACTERIA. 


75 


their  shape,  make  their  outlines  less  distinct,  break  up 
their  arrangement,  and  disturb  them  in  a  variety  of  other 
ways.  Because  of  the  possible  errors  of  appearance  re- 
sulting from  these  causes,  as  well  as  because  it  must  be 
determined  whether  or  not  the  individual  is  motile,  in 
making  a  careful  study  of  a  bacterium  it  must  always  be 
examined  in  the  living,  unstained  condition. 

The  simplest  method  of  making  such  an  examination 
would  be  to  take  a  drop  of  the  liquid,  place  it  upon  a 
slide,  put  on  a  cover,  and  examine. 

While  this  method  is  simple,  it  cannot  be  recommended, 
for  if  the  specimen  should  need  to  be  kept  for  a  time 
much  evaporation  takes  place  at  the  edges  of  the  cover- 
glass,  and  in  the  course  of  an  hour  or  two  has  changed  it 
too  much  for  further  use.  The  immediate  occurrence  of 
evaporation  at  the  edges  also  causes  currents  of  liquid  to 
flow  to  and  fro  beneath  the  cover,  carrying  the  bacteria 
with  them  and  making  it  almost  impossible  to  determine 
whether  the  organisms  under  examination  are  motile  or 
not. 

The  best  way  to  examine  living  micro-organisms  is  in 
what  is  called  the  hanging  drop  (Fig.  5).  A  hollow- 


FIG.  5. — The  "hanging  drop"  seen  from  above  and  in  profile. 

ground  slide  is  used,  and  with  the  aid  of  a  small  camel' s- 
hair  pencil  a  ring  of  vaselin  is  drawn  on  the  slide  about, 
not  in,  the  concavity  at  its  centre.  A  drop  of  the  mate- 
rial to  be  examined  is  placed  in  the  centre  of  a  large 
clean  cover-glass,  and  then  placed  upon  the  slide  so 


76 


PATHOGENIC  BACTERIA. 


that  the  drop  hangs  in,  but  does  not  touch,  the  concavity. 
The  micro-organisms  are  now  hermetically  sealed  in  an 
air-chamber,  and  appear  under  almost  the  same  con- 
ditions as  in  the  cul- 
ture. Such  a  speci- 
men may  be  kept 
from  day  to  day  and 
examined,  the  bac- 
teria continuing  to 
live  until  the  oxygen 
or  nutriment  is  ex- 
hausted. By  means 
of  a  special  appara- 
tus (Fig.  6),  in  which 
the  microscope  is 
stood,  the  growing 
bacteria  may  be 
watched  at  any  tem- 
perature, and  very 
exact  observation  s 
made. 

The  hanging  drop 
should  always  be  ex- 
amined at  the  edge, 
as  the  centre  is  too 
thick. 

In  such  a  specimen 

FiG.  6. — Apparatus  for  keeping  objects  under  it  IS  possible  to  de- 
microscopic  examination  at  constant  tempera-  termilie  the  shape 

size,  grouping,  divis- 
ion, sporulation,  and  motility  of  the  organism  under 
observation. 

Care  should  be  exercised  to  use  a  rather  small  drop, 
especially  for  the  detection  of  motility,  as  a  large  one 
vibrates  very  readily  and  masks  the  motility  of  the 
sluggish  forms. 

When  the  bacteria  to  be  observed  are  in  solid  or  semi- 
solid  culture,  a  small  quantity  of  the  culture  should  be 


METHODS  OF  OBSERVING  BACTERIA.  77 

mixed  up  in  a  drop  of  sterile  bouillon  or  water  and  ex- 
amined. 

In  the  early  days  of  study  efforts  were  made  to  facili- 
tate the  observation  of  bacteria  by  the  use  of  carmin  and 
hematoxylon.  Both  of  these  reagents  tinge  the  proto- 
plasm of  the  organisms  a  little,  but  so  unsatisfactorily 
that  since  Weigert  introduced  the  anilin  dyes  for  the 
purpose  both  of  these  tissue-stains  have  been  rejected. 
The  affinity  between  the  bacteria  and  the  anilin  dyes  is 
peculiar,  and  many  times  is  so  certain  a  reaction  as  to 
become  an  essential  factor  in  the  differentiation  of 
species. 

For  the  study  of  bacteria  in  the  stained  condition  we 
now  employ  the  anilin  dyes  only.  These  wonderful 
colors,  as  numerous  as  the  rainbow  hues,  are  coal-tar 
products.  Hiippe  classifies  them  as  follows  : 

A.   Dyes  prepared  from  anilin  oil. 

1.  Oxidation-products  of  pure  anilin  : 

Methylene  blue, 

Chlorhydrin  blue  (basic  indulin). 

2.  Oxidation-products  of  pure  toluol  : 

Safranin. 

3.  Oxidation-products  of  mixed  anilin  and  toluol  : 

(a)  Rosanilin.     When  pure  this  is  triamido- 

diphenyl-toluyl-karbinol. 

Fuchsin — rosanilin  hydrochlorate.  It  is 
often  mixed  with  the  acetate  and  the 
pararosanilin  acetate  and  hydrochlo- 
rate. The  pure  rosanilin  hydrochlorate 
should  always  be  chosen  for  purposes  of 
staining. 

Azalein  is  rosanilin  nitrite. 

Methylized  and  ethylized  rosanilin  : 
lodin  violet, 
Dahlia, 
lodin  green. 

(b)  Pararosanilin.     The  colorless   pure   para- 

rosanilin is  triamido-triphenyl-karbinol. 


78  PATHOGENIC  BACTERIA. 

Rubin-pararosanilin  hydroclil orate. 
Methylized,     ethylized,     and    benzylized 
pararosanilid  : 

Crystal  violet, 

Gentian  violet, 

Victoria  blue, 

Methyl  green, 

Auramin. 

The  rosanilins  are  more  difficult  to  prepare 
than  the  pararosanilins,  and  are  generally 
mixed  with  them.  The  pararosanilins 
color  more  sharply  than  the  rosanilins. 

4.  Amido-azo  combinations : 

Bismarck  brown, 
Phenylene  brown, 
Vesuvin. 

5.  Chinolin  derivatives : 

Cyanin. 

B.   Naphthalin  group. — Magdala  red. 

The  best  anilin  dyes  made  at  the  present  time,  and 
those  which  have  become  the  standard  for  all  bacterio- 
logical work,  are  made  in  Germany  by  Dr.  Griibler.  In 
ordering  the  stain  the  name  of  this  manufacturer  should 
always  be  specified. 

A  whole  volume  could  easily  be  devoted  to  scientific 
staining.  Indeed,  the  technical  difficulties  encountered 
are  so  great  that  no  explanations  can  be  too  thorough  to 
be  useful.  The  special  methods  essential  for  such  bac- 
teria as  have  peculiar  staining  reactions  will  be  given 
with  the  description  of  the  organism.  General  methods 
only  will  be  discussed  in  this  chapter. 

Cover-glass  Preparations  for  General  Examination. 
— The  material  to  be  examined  must  be  spread  in  the 
thinnest  possible  layer  upon  the  surface  of  a  perfectly 
clean  cover-glass,  and  dried.  Here  it  may  be  remarked 
that  for  bacteriological  purposes  thin  covers  (No.  i)  are 
generally  required,  because  thick' glasses  interfere  with 
the  focussing  of  the  oil-immersion  lenses,  and  that  cover- 


METHODS  OF  OBSERVING  BACTERIA.  79 

glasses  can  never  be  too  clean.  It  is  best  to  immerse 
them  first  in  a  strong  mineral  acid,  then  to  wash  them  in 
water,  then  in  alcohol,  then  in  ether,  and  keep  them  in 
ether  until  they  are  to  be  used.  Except  that  it  some- 
times cracks,  bends,  or  fuses  the  edges  of  the  glasses,  a 
better  and  more  convenient  method  of  cleaning  them  is  to 
wipe  them  as  clean  as  possible,  seize  them  in  fine-pointed 
forceps,  pass  them  repeatedly  through  a  small  Bunsen 
flame  until  it  becomes  greenish  yellow,  then  slowly  ele- 
vate the  glasses  above  the  flame,  so  as  to  allow  them  to 
anneal.  This  maneuvre  removes  the  organic  matter  by 
combustion.  It  is  not  expedient  to  use  covers  twice  for 
bacteriological  work,  though  if  well  cleaned  they  may 
subsequently  be  employed  for  ordinary  microscopic  ob- 
jects. 

To  return  :  After  the  material  spread  upon  the  cover 
has  dried,  it  must  be  fixed  to  the  glass  by  immersion  for 
twenty-four  hours  in  equal  parts  of  absolute  alcohol  and 
ether,  or,  as  is  much  easier  and  more  rapid,  be  passed 
three  times  through  a  flame.  Three  is  not  a  magic 
number,  but  experience  has  shown  that  when  drawn 
through  the  flame  three  times  the  desired  effect  seems 
best  accomplished.  The  Germans  recommend  that  a 
Bunsen  burner  or  a  large  alcohol  lamp  be  used,  that  the 
arm  holding  the  forceps  containing  the  cover-glass  in- 
scribe a  circle  a  foot  in  diameter,  and  that,  as  each  revo- 
lution occupies  a  second  of  time,  the  glass  be  made  to  pass 
through  the  flame  from  apex  to  base  three  times.  This 
is  supposed  to  be  exactly  the  requisite  amount  of  heating. 
The  rule  is  a  good  one  for  the  inexperienced. 

After  fixing,  the  material  is  ready  for  the  stain.  Every 
laboratory  should  be  provided  with  several  stock-solutions 
of  the  more  ordinary  dyes.  These  stock-solutions  are 
satiirated  alcoholic  solutions  made  by  adding  25  grams 
of  the  dye  to  100  c.cm.  of  alcohol!  Of  these  it  is  well  to 
have  fuchsin,  gentian  violet,  and  methylene  blue  always 
made  up.  The  stock-solutions  will  not  stain,  but  are  the 
standards  for  the  manufacture  of  the  working  stains. 


80  PATHOGENIC  BACTERIA. 

For  ordinary  staining  an  aqueous  solution  made  in  a 
simple  manner  is  employed.  A  small  bottle  is  nearly 
filled  with  distilled  water,  and  the  stock-solution  is  added, 
drop  by  drop,  until  the  color  becomes  just  sufficiently  in- 
tense to  prevent  the  ready  recognition  of  objects  through 
it.  Such  a  watery  solution  possesses  the  power  of  readily 
penetrating  the  dried  protoplasm  of  the  bacterium,  taking 
the  stain  with  it.  Alcohol  does  not  have  this  power. 

As  in  the  process  of"  staining  the  cover  is  apt  to  slip 
from  the  fingers  and  spill  the  stain,  it  is  well  to  be  pro- 
vided with  cover-glass  forceps  (Fig.  7),  which  hold  the 


FIG.  7. — Stewart's  cover-glass  forceps. 

glass  in  a  firm  grip  and  allow  of  all  manipulations  with- 
out danger  to  the  fingers  or  clothes.  The  ordinary  in- 
struments are  entirely  unfitted  for  the  purpose,  as  capil- 
lary attraction  draws  the  stain  between  the  blades  and 
makes  certain  the  soiling  of  the  fingers.  Sufficient  stain 
is  allowed  to  run  from  a  pipette  upon  the  smeared  side 
of  the  cover-glass  to  flood  it,  but  not  overflow,  and  is 
allowed  to  remain  for  a  moment  or  two,  after  which  it 
is  thoroughly  washed  off  with  water.  If  the  specimen 
is  one  prepared  for  temporary  use,  it  can  be  examined  at 
once,  mounted  in  a  drop  of  water,  but  under  these  con- 
ditions will  not  appear  as  advantageously  as  if  dried  and 
then  mounted  in  Canada  balsam. 

Sometimes  the  material  to  be  examined  is  too  solid  to 
spread  upon  the  glass  conveniently.  Under  such  circum- 
stances a  drop  of  distilled  water  can  be  added  and  a  minute 
portion  of  the  material  be  mixed  in  it  upon  the  glass. 

The  entire  process  is,  in  brief : 

i.  Spread  the  material  upon  the  cover  ;  2.  Dry — do  not 
heat ;  3.  Pass  three  times  through  the  flame  ;  4.  Stain 


METHODS  OF  OBSERVING  BACTERIA.  8 1 

two  to  three  minutes  ;  5.  Wash  thoroughly  in  water  ; 
6.  Dry  ;  7.  Mount  in  Canada  balsam. 

This  simple  process  suffices  to  stain  most  bacteria. 

Staining  Bacteria  in  Sections  of  Tissue.  —  It  not 
infrequently  happens  that  the  bacteria  to  be  examined 
are  scattered  among  or  enclosed  in  the  cells  of  tissues. 
Their  demonstration  is  then  a  matter  of  some  difficulty, 
and  the  method  employed  is  one  which  must  be  modified 
according  to  the  kind  of  organism  to  be  stained.  Very 
much,  too,  depends  upon  the  preservation  of  the  tissue 
to  be  studied.  As  bacteria  disintegrate  rapidly  in  dead 
tissue,  the  specimen  for  examination  should  be  secured 
as  fresh  as  possible,  cut  into  small  fragments,  and  im- 
mersed in  absolute  alcohol  from  six  to  twenty-four  hours 
to  kill  the  cells  and  bacteria.  Afterward  they  are  re- 
moved from  the  absolute  alcohol  and  kept  in  80-90 
per  cent.,  which  does  not  shrink  the  tissue.  Bichlorid 
of  mercury  may  also  be  used,  but  absolute  alcohol  seems 
to  answer  every  purpose. 

For  ordinary  work  the  following  simple  method  is 
recommended  :  After  the  sections  are  cut,  the  paraffin 
must  be,  and  the  celloidin  would  better  be,  removed. 
From  water  the  sections  are  placed  in  the  same  watery 
stain  used  for  cover-glasses  and  allowed  to  remain  five 
to  eight  minutes.  They  are  next  washed  in  water  for 
several  minutes,  then  decolorized  in  0.5-1  per  cent, 
acetic-acid  solution.  The  acid  removes  the  stain  from 
the  tissues,  and  ultimately  from  the  bacteria  as  well, 
so  that  one  must  watch  carefully,  and  as  soon  as  the 
color  almost  disappears  from  the  sections  remove  them 
to  absolute  alcohol.  At  this  point  the  process  may  be 
interrupted  to  allow  the  tissue-elements  to  be  counter- 
stained  with  alum  carmin  or  any  stain  not  requiring 
acid  for  differentiation,  after  which  the  sections  are 
dehydrated  in  absolute  alcohol,  cleared  in  xylol,  and 
mounted  in  Canada  balsam. 

As  will  be  mentioned  hereafter,  certain  of  the  bacteria 
which  occur  in  tissue  do  not  allow  of  the  ready  penetra- 


82  PATHOGENIC  BACTERIA. 

tion  of  the  color.  For  such  forms  a  more  intense  stain 
must  be  employed.  One  of  the  best  of  these  stains, 
which  can  be  employed  by  the  given  method  both  for 
cover-glasses  and  tissues,  is  Loffler's  alkaline  methylene 
blue: 

Saturated  alcoholic  solution  of  methylene  blue,  30 ; 

i  :  10,000  aqueous  solution  of  caustic  potash,    100. 

Some  bacteria,  as  the  typhoid-fever  bacillus,  decolorize 
so  rapidly  as  to  contraindicate  the  use  of  acid  for  the  dif- 
ferentiation, washing  in  water  or  alcohol  being  sufficient. 
Gram's  Method  of  Staining  Bacteria  in  Tissue. — 
Gram  was  the  fortunate  discoverer  of  a  method  of  stain- 
ing bacteria  in  such  a  manner  as  to  saturate  them  with 
an  insoluble  color.  It  will  be  seen  at  a  glance  what  a 
marked  improvement  this  is  on  the  method  given  above, 
for  now  the  stained  tissue  can  be  washed  thoroughly  in 
either  water  or  alcohol  until  its  cells  are  colorless,  with- 
out fear  that  the  bacteria  will  be  decolorized.  Its  prose- 
cution is  as  follows  :  The  section  is  stained  from  five  to 
ten  minutes  in  a  solution  of  a  basic  anilin  dye — pure 
anilin  (anilin  oil)  and  water.  This  solution,  first  devised 
by  Ehrlich,  is  known  as  Ehrlich's  solution.  The  ordinary 
method  of  preparing  it  is  to  mix  the  following : 

Pure  anilin,  4 ; 

Saturated  alcoholic  solution  of  gentian  violet,     n  ; 

Water,  100. 

Instead  of  gentian  violet,  methyl  violet,  fuchsin,  or  any 
basic  anilin  color  may  be  used.  The  mixture  does  not 
keep  well — in  fact,  seldom  longer  than  six  to  eight  weeks, 
sometimes  not  more  than  two  or  three  ;  therefore  it  is 
best  to  prepare  it  in  very  small  quantity  by  pouring 
about  i  c.cm.  of  pure  anilin  into  a  test-tube,  filling 
the  tube  about  one-half  with  distilled  water,  shaking 
the  mixture  well,  then  filtering  as  much  as  is  desired 
into  a  small  dish.  To  this  the  saturated  alcoholic  solu- 
tion of  the  basic  dye  is  added  until  the  surface  becomes 
distinctly  metallic  in  appearance. 


METHODS  OF  OBSERVING  BACTERIA.  83 

Friedlander  recommends  that  the  section  remain  from 
fifteen  to  thirty  minutes  in  warm  stain,  and  in  many  cases 
the  prolonged  process  gives  better  results. 

From  the  stain  the  section  is  given  a  rather  hasty  wash- 
ing in  water,  and  then  immersed  from  two  to  three  min- 
utes in  Gram's  solution  (a  dilute  Lugol's  solution) : 

lodin  crystals,  i  ; 

Potassium  iodid,  2  ; 

Water,  300. 

While  the  specimen  is  in  the  Gram's  solution  it 
appears  to  turn  a  dark  blackish-brown  color.  When 
removed  from  the  solution  it  is  carefully  washed  in  95 
per  cent,  alcohol  until  no  more  color  is  given  off  and 
the  tissue  assumes  a  grayish  color.  If  it  is  simply 
desired  to  find  the  bacteria,  the  section  is  dehydrated 
in  absolute  alcohol  for  a  moment,  cleared  up  in  xylol, 
and  mounted  in  Canada  balsam.  If  it  is  necessary  to 
study  the  relation  between  the  bacteria  and  the  tissue- 
elements,  a  nuclear  stain,  such  as  alum  carmin  or  Bis- 
marck brown,  may  be  subsequently  used.  Should  a 
nuclear  stain  requiring  acid  for  its  differentiation  be 
desirable,  the  process  of  staining  must  precede  the  Gram 
method  altogether,  so  that  the  acid  shall  not  act  upon 
the  stained  bacteria. 

The  success  of  Gram's  method  rests  upon  the  fact  that 
the  combination  of  mycoprotein,  basic  anilin,  and  the 
iodids  forms  a  compound  insoluble  in  alcohol. 

The  process  described  may  be  summed  up  as  follows  : 

Stain  in  Ehrlich's  anilin-water  gentian  violet  five 
to  thirty  minutes ; 

Wash  momentarily  in  water ; 

Immerse  two  to  three  minutes  in  Gram's  solution ; 

Wash  in  95  per  cent,  alcohol  until  no  more  color 
comes  out ; 

Dehydrate  in  absolute  alcohol ; 

Clear  up  in  xylol ; 

Mount  in  Canada  balsam. 


84  PA  THOGENIC  BA  CTERIA. 

This  method  stains  a  large  variety  of  bacteria  very 
beautifully,  but,  unfortunately,  does  not  stain  them  all, 
and  as  some  of  those  which  do  not  stain  are  important, 
it  seems  well  to  mention  the — 

Spirillum  of  cholera  and  of  chicken-cholera ; 

Bacillus  mallei  (of  glanders) ; 

Bacillus  of  malignant  edema  ; 

Bacillus  pneuinoniae  of  Friedlander ; 

Micrococcus  gonorrhcese  of  Neisser  ; 

Spirochsete  Obermeieri  of  relapsing  fever ; 

Bacillus  of  typhoid  fever  ; 

Bacillus  of  rabbit-septicemia. 

Gram's  method  is  a  method  of  staining  bacteria  in 
tissues,  but  the  fact  that  the  method  colors  some  but  not 
all  bacteria  is  one  of  considerable  importance  from  a  dif- 
ferential point  of  view  ;  and  as  the  difficulty  of  separating 
the  species  of  bacteria  is  so  great  that  every  such  point 
must  be  eagerly  seized  for  assistance,  this  method  be- 
comes one  much  employed  for  cover-glass  preparations, 
where  it  is  more  easily  performed  than  for  sections. 

Gram's  Method  for  Cover-glass  Preparations. — A 
thin  layer  of  the  bacteria  to  be  examined  is  spread  upon 
the  cover-glass,  dried,  and  fixed.  The  cover,  held  in  the 
grip  of  a  cover-glass  forceps,  is  flooded  with  Ehrlich's 
solution.  By  holding  the  cover  flooded  with  stain  over 
a  small  flame  for  a  moment  or  two  the  solution  is  kept 
warm,  and  the  process  of  staining  is  continued  from  two 
to  five  minutes.  If  the  heating  causes  the  stain  to 
evaporate,  more  of  it  must  be  dropped  upon  the  glass, 
so  that  it  does  not  dry  up  and  incrust. 

The  stain  is  poured  off,  and  the  cover  placed  in  a  small 
dish  of  Gram's  solution  and  allowed  to  remain  one-half 
to  two  minutes,  the  solution  being  agitated.  It  is  pos- 
sible to  apply  the  Gram  solution  in  the  same  manner 
in  which  the  stain  is  used,  but  as  a  relatively  larger 
quantity  should  be  employed,  the  dish  seems  preferable. 

The  cover  is  next  washed  in  95  per  cent,  alcohol  until 


METHODS  OF  OBSERVING  BACTERIA.  85 

the  blue  color  is  wholly  or  almost  lost,  after  which  it  can 
be  counter-stained  with  eosin,  Bismarck  brown,  vesuvin, 
etc.,  washed,  dried,  and  mounted  in  Canada  balsam. 
Given  briefly,  the  method  is : 

Stain  with  Khrlich's  solution  two  to  five  minutes  ; 

Gram's  solution  for  one-half  to  two  minutes  ; 

Wash  in  95  per  cent,  alcohol  until  decolorized  ; 

Counter-stain  if  desired  ;  wash  the  counter-stain 
off  with  water ; 

Dry; 

Mount  in  Canada  balsam. 

Method  of  Staining  Spores. — It  has  already  been 
remarked  that  the  peculiar  quality  of  the  spore-capsules 
protects  them  from  the  influence  of  stains  and  disinfect- 
ants to  a  certain  extent.  On  this  account  they  are  much 
more  difficult  to  color  than  the  adult  bacteria.  Several 
methods  are  recommended,  the  one  generally  employed 
being  as  follows :  Spread  the  thinnest  possible  layer  of 
material  upon  a  cover-glass,  dry,  and  fix.  Have  ready 
a  watch-crystalful  of  Ehrlich's  solution,  preferably  made 
of  fuchsin,  and  drop  the  cover-glass,  prepared  side  down, 
upon  the  surface,  where  it  should  float.  Heat  the  stain 
until  it  begins  to  steam,  and  allow  the  specimen  to 
remain  in  the  hot  stain  for  five  to  fifteen  minutes.  The 
cover  is  now  transferred  to  a  3  per  cent,  solution  of  hydro- 
chloric acid  in  water  for  about  one  minute.  Abbott  rec- 
ommends that  the  cover-glass  be  submerged,  prepared 
side  up,  in  a  dish  of  this  solution  and  gently  agitated 
for  exactly  one  minute,  then  removed,  washed  in  water, 
and  counter-stained  with  an  aqueous  solution  of  methyl 
or  methylene  blue. 

In  such  a  specimen  the  spores  should  appear  red,  the 
bacilli  blue. 

I  have  not  generally  found  that  spores  color  so  easily, 
and  for  many  species  the  best  method  seems  to  be  to 
place  the  prepared  cover-glass  in  a  test-tube  half  full  of 
carbol-fuchsin : 


86  PATHOGENIC  BACTERIA. 

Fuchsin,  i  ; 

Alcohol,  10 ; 

5  per  cent,  aqueous  solution  of  phenol  crystals,   100, 

and  boil  it  for  at  least  fifteen  minutes,  after  which  it  is 
decolorized,  either  with  3  per  cent,  hydrochloric  or  2-5  per 
cent,  acetic  acid,  washed  in  water,  and  counter-stained  blue. 

Fiocca  suggests  the  following  rapid  method:  "About 
20  c.cm.  of  a  10  per  cent,  solution  of  ammonium  are 
poured  into  a  watch-glass,  and  10-20  drops  of  a  saturated 
solution  of  gentian  violet,  fuchsin,  methyl  blue,  or  saf- 
ranin  added.  The  solution  is  warmed  until  vapor  begins 
to  rise,  then  is  ready  for  use.  A  very  thinly-spread  cover- 
glass,  carefully  dried  and  fixed,  is  immersed  for  three  to 
five  minutes  (sometimes  ten  to  twenty  minutes),  washed 
in  water,  washed  momentarily  in  a  20  per  cent,  solution 
of  nitric  or  sulphuric  acid,  washed  again  in  water,  then 
counter-stained  with  a  watery  solution  of  vesuvin,  chrys- 
oidin,  methyl  blue,  malachite  green,  or  safranin,  according 
to  the  color  of  the  preceding  stain.  This  whole  process 
is  said  to  take  only  from  eight  to  ten  minutes,  and  to  give 
remarkably  clear  and  beautiful  pictures. ' ' 

Method  of  Staining  Flagella. — This  is  much  more 
difficult  than  the  staining  of  either  the  bacteria  or  their 
spores,  because  each  species  seems  to  behave  differently 
in  its  relation  to  the  stain,  so  that  the  chemistry  of  the 
micro-organismal  products  must  be  taken  into  considera- 
tion. 

The  best  method  introduced  is  that  of  L,6ffler.  In  it 
three  solutions  are  used  : 

A.  A  20  per  cent,  solution  of  tannic  acid,  10  ; 
Cold  saturated  aqueous  solution  of  ferrous  sulphate,    5  ; 
Alcoholic  solution  of  fuchsin  or  methyl  violet,  i. 

B.  A  i  per  cent,  solution  of  caustic  soda. 

C.  An  aqueous  solution  of  sulphuric  acid  of  such  strength 

that  i  c.cm.  will  exactly  neutralize  an  equal  quan- 
tity of  Solution  B. 


METHODS  OF  OBSERVING  BACTERIA.  87 

Some  of  the  bacteria  to  be  stained  are  mixed  upon  a 
cover-glass  with  a  drop  of  distilled  water.  This  is  the 
first  dilution,  but  is  too  rich  in  bacteria  to  allow  the 
flagella  to  show  well,  so  that  it  is  recommended  to  prepare 
a  second  dilution  by  placing  a  small  drop  of  distilled 
water  upon  a  cover  and  taking  a  small  portion  from  the 
first  cover  to  the  second,  spreading  it  over  the  entire  sur* 
face.  The  material  is  allowed  to  dry,  and  is  then  fixed 
by  passing  it  three  times  through  the  flame.  When  this 
is  done  with  forceps  there  is  some  danger  of  the  prepara- 
tion becoming  too  hot,  so  Loffler  recommends  that  the 
glass  be  held  in  the  fingers  while  the  passes  through  the 
flame  are  made. 

The  cover-glass  is  now  held  in  forceps,  and  the  mordant, 
Solution  A,  is  dropped  upon  it  until  it  is  well  covered. 
The  cover  is  wanned  until  it  begins  to  steam,  and  the 
stain  replaced  as  it  evaporates.  It  must  not  be  heated  too 
strongly  ;  above  all  things,  must  not  boil.  This  solution 
is  allowed  to  act  from  one-half  to  one  minute,  is  then 
washed  in  distilled  water,  then  in  absolute  alcohol  until  all 
traces  of  the  solution  have  been  removed.  The  real  stain 
— Loffler  recommends  an  anilin-water  fuchsin  (Bhrlich's 
solution) — which  should  have  a  neutral  reaction,  is  now 
dropped  on  so  as  to  cover  the  specimen,  and  heated  for  a 
minute  until  vapor  begins  to  arise;  it  is  then  washed  off 
carefully,  dried,  and  mounted  in  Canada  balsam.  To 
obtain  this  neutral  reaction  enough  of  the  i  per  cent, 
sodium-hydrate  solution  is  added  to  an  amount  of  the 
anilin-water-fuchsin  solution  having  a  thickness  of  sev- 
eral centimeters  to  begin  to  change  the  transparent  inrf> 
an  opaque  solution.  Such  a  specimen  may  or  may  not 
show  the  flagella.  If  not,  before  proceeding  farther  it  is 
necessary  to  study  the  products  of  the  bacterium  in  cul- 
ture-media. If  by  its  growth  the  organism  elaborates 
alkalies,  Solution  C,  in  proportion  from  i  drop  to  i  c.cm. 
in  1 6  c.cm.  of  the  mordant  A,  must  be  added,  and  the 
process  repeated  again  and  again  until  the  proper  amount 
is  determined.  On  the  other  hand,  if  the  organism  by 


88  PATHOGENIC  BACTERIA. 

its  growth  produces  acid,  Solution  B  must  be  added, 
drop  by  drop,  until  i  in  16  cm.  have  been  attained,  and 
numerous  experiments  made  to  see  when  the  flagella 
will  appear.  lyoffler  lias  fortunately  worked  out  the 
amounts  required  for  some  of  the  species,  and  of  the 
more  important  ones  the  following  amounts  of  Solutions 
B  and  C  must  be  added  to  16  c.cm.  of  Solution  A  to 
attain  the  desired  effect : 

Cholera  spirillum,  ^-i  drop  of  Solution  C ; 

Typhoid  fever,  i  c.cm.  of  Solution  B  ; 

Bacillus  subtilis,  28-30  drops  of  Solution  B  ; 

Bacillus  of  malignant  edema,  36-37  drops  of  Solution  B. 

Part  of  the  success  of  the  staining  depends  upon 
having  the  bacteria  thinly  spread  upon  the  glass,  and  as 
free  from  albuminous  and  gelatinous  materials  as  possi- 
ble. The  cover-glass  must  be  cleaned  most  painstakingly  : 
too  much  heating  in  fixing  must  be  avoided.  After  using 
and  washing  off  the  mordant,  the  preparation  should  be 
dried  before  the  application  of  the  anilin-water-fuchsin 
solution. 

Bunge  suggests  a  mordant  consisting  of  a  concentrated 
aqueous  tannin  solution  and  a  i  :  20  solution  of  liq.  ferri 
sesquichloridi  in  water.  The  best  mixture  seems  to  be 
3  parts  of  the  tannin  solution  to  i  part  of  the  diluted 
iron  solution.  To  10  c.cm.  of  this  mixture  i  c.cm.  of  a 
concentrated  aqueous  fuchsin  solution  is  added.  It  is 
not  necessary  to  prepare  this  mordant  fresh  for  each 
staining,  as  it  seems  to  improve  with  age.  The  use  of 
acid  and  alkaline  solutions  added  to  the  mordant  is  dis- 
pensed with. 

The  bacteria  are  carefully  fixed  to  the  glass,  stained 
with  the  mordant  for  five  minutes,  warming  a  little  to- 
ward the  end,  washed,  dried,  and  subsequently  colored 
with  carbol-fuchsin  warmed  a  little. 

Bacteria  can  best  be  measured  by  an  eye-piece  microm- 
eter. As  these  instruments  vary  somewhat  in  con- 


METHODS  OF  OBSERVING  BACTERIA.  89 

struction,  the  unit  of  measurement  for  each  objective 
magnification  or  the  method  of  manipulating  the  adjusta- 
ble instruments  must  be  learned  from  dealers'  catalogues. 
Photographing  bacteria  requires  special  apparatus  and 
methods,  which  are  fully  described  in  text-books  upon 
the  subject. 


CHAPTER  V. 

STERILIZATION  AND  DISINFECTION. 

BEFORE  considering  the  cultivation  of  bacteria  and 
the  preparation  of  media  for  that  purpose  it  is  necessary 
to  discuss  methods  of  destroying  bacteria  whose  acci- 
dental presence  might  ruin  our  experiments. 

The  dust  of  the  atmosphere,  as  has  already  been  shown, 
is  almost  constant  in  its  micro-organismal  contamination, 
so  that  the  spores  of  moulds  and  bacilli,  together  with 
yeasts  and  micrococci,  constantly  settle  from  it  upon  our 
glassware,  enter  our  pots,  kettles,  funnels,  etc.,  and  would 
ruin  every  culture-medium  with  which  we  operate  did 
we  not  take  measures  for  their  destruction. 

Micro-organisms  may  be  killed  by  heat  or  by  the  action 
of  chemicals,  the  processes  being  generically  termed  dis- 
infection. The  destruction  of  the  germs  by  heat  is  gen- 
erally called  sterilization.  A  chemical  agent  causing  the 
death  of  bacteria  is  called  a  germicide.  An  object  which 
is  entirely  free  from  bacteria  and  their  spores  is  described 
as  sterile.  Certain  substances  whose  action  is  detrimental 
to  the  vitality  of  bacteria  and  prevents  their  growth  amid 
otherwise  suitable  surroundings  are  termed  antiseptics. 

The  study  of  sterilization,  disinfection,  and  antisepsis 
will  naturally  lead  us  through  the  following  subdivisions  : 

I.  The  sterilization  and  protection  of  instruments  and 
glassware  used  in  experimentation. 

II.  The  sterilization  and  protection  of  culture-media. 

III.  The  disinfection  of  the  instruments,  ligatures,  etc. 
and  the  hands  of  the  surgeon,  and  the  use  of  antiseptics. 

IV.  The  disinfection  of  sick-chambers  and  their  con- 
tents, as  well  as  the  dejecta  and  discharges  of  patients 
suffering  from  contagious  and  infectious  diseases. 

90 


STERILIZATION  AND  DISINFECTION.  91 

The  Sterilization  and  Protection  of  Instruments 
and  Glassware  Used  in  Experimentation. — Steriliza- 
tion may  be  accomplished  by  either  moist  or  dry  heat. 
For  the  perfect  sterilization  of  objects  capable  of  with- 
standing it  dry  heat  is  preferable,  because  more  certain 
in  its  action.  If  we  knew  just  what  organisms  we  had 
to  deal  with,  we  might  be  able  in  many  cases  to  save 
time  and  gas,  but  while  some  simple  non-spore-producing 
forms  are  killed  at  a  temperature  of  60°  C.,  others  can 
withstand  boiling  for  an  hour  ;  it  is  therefore  best  to 
employ  a  temperature  high  enough  to  kill  all  with  cer- 
tainty. Platinum  wires  used  for  inoculation  are  held  in 
the  direct  flame  until  they  become  incandescent.  In 
sterilizing  such  wires  attention  must  be  bestowed  upon 
the  glass  handle,  which  should  be  held  in  the  flame  for 
at  least  half  its  length  for  a  few  moments  when  used  for 
the  first  time  each  day.  Carelessness  in  this  respect  may 
cause  the  loss  of  much  time  by  contaminating  cultures. 

Knives,  scissors,  and  forceps  may  be  exposed  for  a  very 
brief  time  to  the  direct  flame,  but  this  affects  the  temper 
of  the  steel  when  continued  too  long.  They  may  also 
be  boiled,  steamed,  or  carbolized. 

All  glassware  is  sterilized  by  exposure  to  a  sufficiently 
high  temperature,  150°  C.  or  302°  F.,  for  one  hour  in  the 
well-known  hot-air  closet  (Fig.  8).  A  temperature  of 
150°  C.  is  sufficient  to  kill  all  known  bacteria  and  their 
spores  if  continued  for  an  hour. 

Rubber  stoppers,  corks,  wooden  apparatus,  and  other 
objects  which  are  warped,  cracked,  charred,  or  melted 
by  so  high  a  temperature  must  be  sterilized  by  moist 
heat  in  the  steam  apparatus  for  at  least  an  hour  before 
they  can  be  pronounced  sterile. 

It  must  always  be  borne  in  mind  that  after  sterilization 
has  been  accomplished  the  same  sources  of  contamination 
that  originally  existed  are  still  present,  and  begin  to 
operate  as  soon  as  the  objects  are  removed  from  the 
sterilizing  apparatus. 

To  Schroder   and  Van  Dusch   belong   the   credit   of 


92  PATHOGENIC  BACTERIA. 

having  first  shown  that  when  the  months  of  flasks  and 
tubes  are  closed  with  plugs  of  sterile  cotton  no  germs 
can  filter  through.  This  observation  has  been  of  ines- 
timable value  to  every  bacteriologist.  Before  sterilizing 


FIG.  8. — Hot-air  sterilizer. 

flasks  and  tubes'  we  plug  them  with  ordinary  raw  cotton, 
and  are  sure  that  afterward  their  interiors  will  remain 
free  from  the  access  of  germs  until  opened.  Instruments 
may  be  sterilized  wrapped  in  cotton,  to  be  opened  only 
when  ready  for  use ;  or  instruments  and  rubber  goods 
sterilized  by  steam  can  subsequently  be  wrapped  in 
sterile  cotton  and  kept  for  use.  It  is  of  the  utmost 
importance  to  carefully  protect  every  sterilized  object, 
and  to  allow  as  little  dust  to  collect  upon  it  as  possible, 
in  order  that  the  object  of  the  sterilization  be  not  de- 
feated. As  the  spores  of  moulds  falling  upon  cotton 
sometimes  grow  and  allow  their  mycelia  to  work  their 
way  through  and  drop  into  a  culture-medium,  Roux 


STERILIZATION  AND  DISINFECTION. 


93 


has  introduced  little  paper  caps  with  which  the  cotton 
stoppers  are  protected  from  the  dust.  These  are  easily 
made  by  curling  a  small  square  of  paper  into  a  "cornu- 
copia, ' '  fastening  by  turning  up  the  edge  or  putting  in  a  pin. 
The  paper  is  placed  over  the  stopper  before  the  sterilization, 
after  which  no  contamination  of  the  cotton  can  occur. 

Sterilization  of  Culture-media. — As  almost  all  of  the 
culture-media  contain  about  80  per  cent,  of  water,  which 
would  be  evaporated  in  the  hot-air  closet,  so  that  the 
material  would  be  destroyed,  hot-air  sterilization  is  not 
appropriate  for  them.  Sterilization  by  streaming  steam 
is  the  best  and  surest  method.  The  prepared  media  are 

placed  in  flasks  or  tubes  care- 
fully plugged  with  cotton  and 
previously  sterilized  with  dry 
heat,  and  then  sterilized  in  what 
is  known  as  Koch's  steam  appa- 
ratus (Fig.  9)  or  in  Arnold's 


FIG.  9. — Koch's  steam  sterilizer. 


FIG.  10. — Arnold's  steam  sterilizer. 


steam  sterilizer  (Fig.  10),  which  is  more  convenient  and 
more  generally  useful. 

The  temperature  of  boiling  water,   100°  C.,  does  not 


94 


PATHOGENIC  BACTERIA. 


kill  many  spores,  so  that  the  exposure  of  culture-media 
to  streaming  steam  is  of  little  use  unless  applied  in 
a  systematic  manner — intermittent  sterilization — based 
upon  a  knowledge  of  sporulation. 

In  carrying  out  the  intermittent  sterilization  the  cul- 
ture-medium is  exposed  for  fifteen  minutes  to  the  passage 
of  streaming  steam  in  the  apparatus  or  to  some  tem- 
perature judged  to  be  sufficiently  high,  so  that  the  bac- 
teria contained  in  it  are  killed.  As  the  spores  remain 
uninjured,  the  medium  is  stood  aside  in  a  cool  place  for 
twenty-four  hours,  and  the  spores  allowed  to  develop  into 
perfect  bacteria. 

When  the  twenty-four  hours  have  passed  the  culture- 
medium  is  again  placed  in  the  apparatus  and  exposed  to 

the  same  temperature, 
until  these  newly-devel- 
oped bacteria  are  also 
killed.  Eventually, 
the  process  is  repeated 
a  third  time,  lest  a  few 
spores  remain  alive  and 
capable  of  spoiling  the 
material.  When  prop- 
erly sterilized  in  this 
way,  culture- media  will 
remain  free  from  con- 
tamination until  time 
and  evaporation  cause 
them  to  dry  up.  If  her- 
metically sealed,  a  ster- 
ile medium  will  keep 
indefinitely. 

If  it  should  be  neces- 
sary to  sterilize  culture- 

FIG.  n.-Autoclave  for  rapid  sterilization  media  ^  Q  not  wait_ 

by  superheated  steam  under    pressure. 

ing  the  three  days  re- 
quired by  the  intermittent  method,  it  may  be  done  by 
superheated  steam  in  an  autoclave  (Fig.  n).  Here  under 


STERILIZA  TION  AND  DISINFECTION. 


95 


a  pressure  of  two  or  three  atmospheres  sufficient  heat  is 
generated  to  destroy  the  spores.  The  objections  to  this 
method  are  that  it  sometimes  turns  the  agar-agar  dark, 
and  that  it  is  likely  to  destroy  the  gelatinizing  power  of 
the  gelatin,  which  after  sterilization  remains  liquid. 


FIG.  12. — Pasteur-Chamberland  filter  arranged  to  filter  under  pressure. 

lyiquids  may  also  be  sterilized  by  filtration — i.  e.  by 
passing  them  through  unglazed  porcelain  or  some  other 
material  whose  interstices  are  sufficiently  fine  to  resist  the 
passage  of  the  bacteria.  This  method  is  largely  employed 


96 


PATHOGENIC  BACTERIA. 


for  the  sterilization  of  the  unstable  toxins  and  anti- 
toxins, which  are  destroyed  by  heat.  Various  substances 
have  been  used  for  filtration,  as  stone,  sand,  powdered 
glass,  etc.,  but  experimentation  has  shown  porcelain  to 
be  the  only  reliable  filter  against  bacteria.  Even  this 
material,  whose  interstices  are  so  small  as  to  allow  the 
liquid  to  pass  through  with  great  slowness,  is  only  cer- 
tain in  its  action  for  a  time,  for  after  it  has  been  used 
considerably  the  bacteria  seem  able  to  work  their  way 
through.  To  be  certain  of  the  efficacy  of  such  a  filter 
the  fluid  first  passed  through  must  be  tested  by  cultiva- 
tion methods.  The  complicated  Pasteur-Chamberland 
and  the  simple  Kitasato  and  Reichel  filters  are  shown  in 
Figures  12,  13,  and  14. 

After  having  been  used  a  porcelain  filter  must  be  dis- 
infected, scrubbed,  dried  thoroughly,  and  then  heated  in 
a  Bunsen  burner  or  blowpipe  flame  until  all  the  organic 


FIG.  13. — Kitasato's  filter:  a,  por- 
celain bougie  ;  b,  attachment  for  suc- 
tion-pump; c,  reservoir;  d,  sterile 
receiver. 


FIG.  14. — Reichel's  bacteriologic  filter 
of  unglazed  porcelain :  A,  sterile  re- 
ceiver; B,  porcelain  filter;  c,  d,  attach- 
ments for  pump. 


matter  is  consumed.     In  this  firing  process  the  filter  first 
turns  black  as  the  organic  matter  chars,   then  becomes 


STERILIZATION  AND  DISINFECTION. 


97 


white  as  it  is  consumed.  The  greatest  care  must  be 
exercised  in  cleansing,  and  especially  must  care  be  taken 
that  the  porcelain  is  dry  before  entering  the  fire,  as  it 
will  certainly  crack  if  moist. 

Before  using  a  new  filter  it  should  be  sterilized  by  dry 
heat,  then  connected  with  receivers  and  tubes,  also  care- 
fully sterilized.  It  should  not  be  forgotten  that  the  fil- 
tered material  is  still  a  good  culture-medium  and  must  be 
handled  with  the  greatest  care. 

While  the  filtration  of  water,  peptone  solution,  and 
bouillon  is  comparatively  easy,  gelatin  and  blood-serum 
pass  through  with  great  difficulty,  and  speedily  gum  the 
filter,  so  that  it  is  useless  until  fired. 

A  convenient  apparatus  used  by  the  author  for  the  rapid 
filtration  of  large  quantities  is  shown  in  the  accompany- 
ing illustration  (Fig.  15). 


FIG.  15. — Apparatus  for  the  rapid  filtration  of  toxins,  etc. 

The  Disinfection  of  Instruments,  Ligatures,  Sutures, 
the  Hands,  etc. — There  are  certain  objects  used  by  the 
surgeon  which  cannot  well  be  rendered  incandescent, 
exposed  to  dry  heat  at  150°  C.,  steamed,  or  intermittently 
heated  without  injury.  For  these  objects  disinfection 
must  be  practised.  Ever  since  Sir  Joseph  Lister  intro- 
duced antisepsis,  or  disinfection,  into  surgery  there  has 
been  a  struggle  for  the  supremacy  of  this  or  that  highly- 
recommended  germicidal  substance,  with  two  results — 
viz.  that  a  great  number  of  feeble  germicides  have  been 
discovered,  and  that  belief  in  the  efficacy  of  all  germi- 
cides has  been  somewhat  shaken;  hence  the  origin  of  the 

7 


98  PATHOGENIC  BACTERIA. 

successful  aseptic  surgery  of  the  present  day,  which 
strives  to  prevent  the  entrance  of  germs  into,  rather  than 
their  destruction  after  admission  to,  the  wound. 

For  a  complete  discussion  of  the  subject  of  antiseptics 
in  relation  to  surgery  the  reader  must  be  referred  to  the 
large  text-books  of  surgery,  where  much  space  is  thus 
occupied.  A  short  list  of  useful  germicides  of  which 
the  respective  values  are  given,  and  a  brief  discussion 
of  some  of  the  more  important  measures,  can  alone  find 
space  in  these  pages.  The  antiseptic  value  of  some  of 
the  principal  substances  used  may  be  expressed  as  fol- 
lows, the  figures  indicating  the  strength  of  the  solution 
necessary  to  prevent  the  development  of  bacteria  : 

Pyoktanin  (methyl  violet)  .  i  :  2,000,000 — i  15000. 
Bichlorid  of  mercury    .    .    .  i :  14,300. 

Hydrogen  peroxid i :  20,000. 

Formalin i :  20,000. 

Nitrate  of  silver  .   ....    .1:12,500. 

Creolin i :  5000  to  i  :  200  (does  not  kill 

anthrax). 
Chromic  acid i :  5000. 


Thymol 

Salicylic  acid 

Potassium  bichromate  .    . 

Zinc  chlorid 

Potassium  permanganate 


:  1340. 
:  looo. 
1909. 


:  285  ;  not  prompt  in  action  . 


Nitrate  of  lead i :  277. 

Boracic  acid i :  143. 

Chloral  hydrate i :  107. 

Ferrous  sulphate i :  90 — i  :  200,  Sternberg. 

Calcium  chlorid i :  25. 

Creosote        i :  20. 

Carbolic  acid i :  20  : :  i  :  50. 

Alcohol  .    .    , i :  10. 

Ether.     Pure  ether  will  not  kill  anthrax  spores  immersed 
in  it  for  eight  days. 

The  value  of  antiseptics,  like  that  of  disinfectants,  is 
always  relative,  the  destructive  as  well  as  the  inhibitory 
power  of  the  solution  varying  with  the  micro-organism 
upon  which  it  acts.  The  following  table,  from  Boer, 
will  illustrate  this : 


STERILIZA  TION  AND  DISINFECTION,  99 

Methyl  Violet  (Pyoktanin}. 

Restrains.  Kills. 

Anthrax  bacillus i :  70,000  i  :  5000 

Diphtheria i :  10,000  i :  2000 

Glanders .  i :  2500  i :  150 

Typhoid .  i :  2500  i :  150 

Cholera  spirillum i :  30,000  i :  1000 

Large  numbers  of  both  strongly  and  feebly  antiseptic 
substances  have  purposely  been  omitted  from  the  above 
lists,  compiled  from  Sternberg  and  Micquel,  as  either  in- 
appropriate for  ordinary  use  or  as  having  been  replaced 
by  better  agents. 

The  disinfection  of  the  skin,  both  the  hands  of  the 
surgeon  and  the  part  about  to  be  incised,  is  a  matter  of 
importance.  It  is  almost  impossible  to  secure  absolute 
sterility  of  the  hands,  so  deeply  do  the  skin-cocci  pene- 
trate between  the  layers  of  the  scarf-skin.  The  method  at 
present  generally  employed,  and  recommended  by  Welch 
and  Hunter  Robb,  is  as  follows :  The  nails  must  be 
trimmed  short  and  perfectly  cleansed.  The  hands  are 
washed  thoroughly  for  ten  minutes  in  water  of  as  high  a 
temperature  as  can  comfortably  be  borne,  soap  and  a  brush 
previously  sterilized  being  freely  used,  and  afterward  the 
excess  of  soap  washed  off  in  clean  hot  water.  The  hands 
are  then  immersed  for  from  one  to  two  minutes  in  a 
warm  saturated  solution  of  permanganate  of  potassium, 
then  in  a  warm  saturated  solution  of  oxalic  acid,  until 
complete  decolorization  of  the  permanganate  occurs,  after 
which  they  are  washed  free  from  the  acid  in  clean  warm 
water  or  salt-solution.  Finally,  they  are  soaked  for  two 
minutes  in  a  i  :  500  solution  of  bichlorid  of  mercury, 
after  which  they  are  ready  for  use. 

Surgical  dressings  are  generally  sterilized  by  super- 
heated steam,  which,  as  has  been  shown,  destroys  all 
germs.  Ligatures  and  sutures  of  silk,  gut,  chromicized 
gut,  silkworm  gut,  etc.,  having  been  boiled,  are  kept 
either  in  alcohol  or  in  an  alcoholic  solution  of  bichlorid 


100  PATHOGENIC  BACTERIA, 

of  mercury,  or,  if  this  causes  them  to  become  too  brittle, 
in  a  watery  solution  of  bichlorid. 

At  present,  in  most  hospitals,  instruments  are  boiled 
before  using,  and  during  the  operation  are  either  kept  in 
the  boiled  water  or  in  carbolic  solution. 

During  the  operation  the  wound  is  frequently  to  be 
washed  with  carbolic  solution  or  bichlorid  of  mercury, 
i :  2000,  applied  by  sterile  sponges.  To  La  Place  belongs 
the  credit  of  observing  that  the  efficacy  of  these  germi- 
cides is  greatly  increased  by  the  addition  of  a  small 
amount  of  acid,  by  which  their  penetration  is  increased 
and  the  formation  of  insoluble  albuminates  lessened. 

The  knowledge  that  the  action  of  germicides  is  chem- 
ical, and  that  the  destruction  of  the  bacteria  is  due  to  the 
combination  of  the  germicide  with  the  mycoprotein,  is 
apt  to  lessen  our  confidence  in  the  permanence  of  their 
action.  Geppert  has  shown  of  bichlorid  of  mercury  that 
in  the  reaction  between  it  and  anthrax  spores  the  vitality 
of  the  latter  seems  lost,  but  that  the  precipitation  of  the 
bichlorid  from  this  combination  by  the  action  of  ammo- 
nium sulphid  restores  the  vitality  of  the  spore. 

Again,  the  fact  that  some  of  the  antiseptics,  as  nitrate 
of  silver  and  bichlorid  of  mercury,  are  at  once  precipi- 
tated by  albumins,  and  thus  lose  their  germicidal  and 
antiseptic  powers,  limits  the  scope  of  their  employment. 
I  think  it  may  be  safely  said  that  carbolic  acid  is  the 
most  reliable  and  most  generally  useful  of  all  the  germi- 
cides and  antiseptics. 

The  Disinfection  of  Sick-chambers,  Dejecta,  etc. — 
What  has  just  been  remarked  concerning  the  unreliability 
of  many  of  the  germicidal  substances  is  eminently  a 
propos  of  the  disinfection  of  dejecta.  It  is  useless  to 
mix  bichlorid  of  mercury  with  typhoid  stools  or  tubercu- 
lar sputum  rich  in  albumin,  and  imagine  these  substances 
rendered  harmless  in  consequence.  It  should  not  be  for- 
gotten that  the  sick  patient  is  less  the  means  of  convey- 
ing the  contagium  than  the  objects  with  which  he  is  in 
contact,  which  can  be  carried  to  other  rooms  or  houses 


STERILIZA  TION  AND  DISINFECTION.         IOI 

during  or  after  the  progress  of  the  disease.  A  careful 
consideration  of  the  condition  of  the  sick-room  will 
lead  us  to  a  clear  understanding  of  its  bacteriological 
condition. 

The  Air  of  the  Sick-room. — It  is  impossible  to  sterilize 
or  disinfect  the  atmosphere  of  a  room  during  its  occu- 
pancy by  the  patient,  or  in  all  probability  after  his  exitus 
from  it.  The  concentration  of  the  disinfecting  solutions 
given  above  must  make  obvious  the  foolishness  of  placing 
beneath  the  bed  or  in  the  corners  of  a  room  small  recep- 
tacles filled  with  carbolic  acid  or  chlorinated  lime.  These 
can  serve  no  purpose  for  good,  and  may  be  potent  for 
harm  by  obscuring  the  disagreeable  odors  emanating 
from  materials  which  should  be  removed  from  the  room 
by  the  still  more  disagreeable  odors  of  the  disinfectants. 
The  practice  of  such  a  custom  is  only  comparable  to  the 
old  faith  in  the  virtue  of  asafetida  tied  up  in  a  corner  of 
the  handkerchief  as  a  preventive  of  cholera  and  small- 
pox. 

During  the  period  of  illness  a  chamber  in  which  the 
patient  is  confined  should  be  freely  ventilated,  so  that  its 
atmosphere  is  constantly  changing  and  replacing  the 
closeness  so  universally  an  accompaniment  of  fever  by 
fresh,  pure  air — a  comfort  to  the  patient  and  a  protection 
to  the  doctors  and  nurses. 

After  recovery  or  death  one  should  rely  less  upon  fu- 
migation than  upon  the  disinfection  of  the  walls  and 
floor,  the  similar  disinfection  of  the  wooden  part  of  the 
furniture,  and  the  sterilization  of  all  else.  The  fumes 
of  sulphur  may  do  some  good — when  combined  with 
steam,  much  good — but  are  greatly  overestimated,  and 
disinfection  combined  with  fresh  air  and  sunlight  is 
much  better. 

Indeed,  I  would  recommend  that  after  opening  the  doors 
and  windows  of  the  sick-chamber  the  atmosphere  of  the 
whole  house  be  forgotten  and  attention  be  devoted  to 
other  things. 

The  Dejecta. — A  little  thought  will  direct  attention  to 


102  PATHOGENIC  BACTERIA. 

those  of  the  dejections  which  are  dangerous  to  the  com- 
munity and  promote  efforts  for  their  complete  steriliza- 
tion. In  cases  of  diphtheria  the  vomit,  expectorations, 
and  nasal  discharges  are  most  important.  They  should 
be  received  in  old  rags  or  in  Japanese  paper  napkins — 
not  handkerchiefs  or  towels — and  should  be  burned.  The 
sputum  of  tuberculous  patients  should  either  be  collected 
in  a  glazed  earthen  vessel  which  can  be  subjected  to  boil- 
ing and  disinfection,  or,  as  is  an  excellent  plan,  should  be 
received  in  Japanese  rice-paper  napkins,  which  can  at 
once  be  burned.  These  napkins  are  not  quite  as  good 
as  the  small  pasteboard  boxes  (Fig.  16)  recommended  by 


FIG.  1 6. — Pasteboard  cup  for  receiving   infectious   sputum.     When  used  the 
pasteboard  can  be  removed  from  the  iron  frame  and  burned. 

some  city  boards  of  health,  because,  being  highly  absorb- 
ent, the  sputum  is  apt  to  soak  through  and  soil  the  rin- 
gers, etc.  Tuberculous  patients  should  be  provided  with 
rice-paper  instead  of  handkerchiefs,  and  should  have  their 
towels,  knives,  forks,  spoons,  plates,  etc.  kept  strictly 
apart  from  the  others  of  the  household  (though  the  pa- 
tients, whose  mental  acuity  makes  their  sensibilities  very 
pronounced,  need  never  be  told  of  their  isolation),  and 
frequently  boiled  for  considerable  lengths  of  time. 

The  excreta  from  typhoid-fever  and  cholera  cases  re- 
quire particular  attention.  These,  and  indeed  all  alvine 
matter  possibly  the  source  of  infection  or  contagion, 
should  be  received  in  glazed  earthen  vessels  and  imme- 
diately intimately  mixed  with  a  5  per  cent,  solution 
of  chlorinated  lime  (containing  25  per  cent,  of  chlorin) 
if  semi-solid,  or  with  the  powder  if  liquid,  and  allowed 


STERILIZATION  AND  DISINFECTION.         103 

to  stand  for  an  hour  before  being  thrown  into  the 
drain. 

The  Clothing,  etc. — All  bed-clothing  which  has  been 
used  in  the  sick-room,  all  towels,  napkins,  handkerchiefs, 
night-robes,  underclothes,  etc.  which  have  been  used  by 
the  sick,  and  all  towels,  napkins,  handkerchiefs,  caps, 
aprons,  and  outside  dresses  worn  by  the  nurse,  should  be 
regarded  as  infected  and  subjected  to  sterilization.  The 
only  satisfactory  method  of  doing  this  is  by  prolonged 
subjection  to  steam  in  a  special  apparatus ;  but,  as  this 
is  only  possible  in  hospitals,  the  next  best  thing  is  boiling 
for  some  time  in  the  ordinary  wash-boiler.  When  pos- 
sible, the  clothes  should  be  soaked  in  i  :  2000  bichlorid 
solution  before  or  after  boiling,  and  in  drying  should 
hang  in  the  sun  and  wind.  Woollen  underwear  can  be 
treated  exactly  as  if  of  cotton.  The  woollen  clothing  of 
the  patient,  if  infected,  requires  special  treatment.  For- 
tunately, the  infection  of  the  outer  woollen  garments  is 
unusual.  The  only  reliable  method  for  their  purification 
is  prolonged  exposure  to  hot  air  at  110°  C.  In  private 
practice  it  becomes  a  grave  question  what  shall  be  done 
with  these  articles.  Prolonged  exposure  to  fresh  air  and 
sunlight  will  aid  in  rendering  them  harmless  ;  when  it 
is  certain  that  articles  of  wool  are  infected,  they  may  be 
sent  to  the  city  hospital  or  to  certain  of  the  moth-destroy- 
ing and  fumigating  establishments  which  can  be  found 
in  all  large  cities,  and  be  baked. 

The  Furniture,  etc. — The  wholesale  destruction  of  fur- 
niture practised  in  earlier  times  has  at  present  become 
unnecessary.  The  doctor,  if  he  properly  performs  his 
functions,  will  save  much  trouble  and  money  for  his 
patient  by  ordering  the  immediate  isolation  of  his  charge 
in  an  uncarpeted,  scantily-  and  cheaply-furnished  room 
the  moment  an  infectious  disease  is  suspected,  before 
much  infection  can  have  occurred.  However,  if  before 
his  removal  the  patient  has  occupied  another  bed,  its 
clothing  should  be  promptly  handled  in  the  above- 
described  manner. 


104  PATHOGENIC  BACTERIA. 

After  the  illness  the  walls  of  the  rooms,  including  the 
ceiling,  may  be  rubbed  with  fresh  bread,  which  L,6ffler 
has  shown  to  be  efficacious  in  collecting  the  bacteria, 
or,  if  possible,  should  be  whitewashed.  If  the  walls  are 
hung  with  paper,  it  should  be  dampened  with  i :  1000 
bichlorid-of-mercury  solution  before  new  paper  is  hung. 
The  floor  should  be  scoured  with  5  per  cent,  carbolic- 
acid  solution  or  i :  1000  bichlorid  of  mercury,  and  all  the 
wooden  articles  wiped  off  two  or  three  times  with  the 
same  solution  employed  for  the  floor.  In  this  scouring 
no  soap  can  be  used,  as  it  destroys  the  virtue  of  the 
germicide.  If  a  straw  mattress  was  used,  it  should  be 
burned  and  the  cover  boiled.  If  a  hair  mattress  was 
used,  it  can  be  steamed  or  baked  by  the  manufacturers, 
who  generally  have  ovens  for  the  purpose.  Curtains, 
shades,  etc.  should  receive  proper  attention,  but  of  course 
the  greater  the  precautions  exercised  in  the  beginning, 
the  fewer  the  articles  which  will  need  attention  in  the 
end.  They  should  be  removed  before  the  case  has 
developed. 

The  patient,  whether  he  lives  or  dies,  may  also  be 
a  means  of  spreading  the  disease  unless  specially  cared 
for.  After  convalescence  the  body  should  be  bathed  with 
a  weak  bichlorid-of-mercury  solution  or  with  a  2  per 
cent,  carbolic-acid  solution,  or  with  25-50  per  cent,  alco- 
hol, before  the  patient  is  allowed  to  mingle  with  society, 
and  the  hair  should  either  be  cut  off  or  carefully  washed 
with  the  above  solution.  In  desquamative  diseases  it 
seems  best  to  have  the  entire  body  anointed  with  cos- 
molin  once  daily,  the  unguent  being  well  rubbed  in,  in 
order  to  prevent  the  particles  of  epidermis  being  distrib- 
uted through  the  atmosphere.  Carbolated  cosmolin  may 
be  better  than  the  plain,  not  because  of  the  very  slight 
antiseptic  value  it  possesses,  but  because  it  helps  to  allay 
the  itching  which  may  be  part  of  the  desquamative 
process. 

After  the  patient  is  about  the  room  again,  common 
sense  will  prevent  the  admission  of  strangers  until  all 


STERILIZATION  AND  DISINFECTION.         105 

the  disinfective  measures  have  been  adopted,  and  after 
this,  touching,  and  especially  kissing  him,  should  be 
omitted  for  some  time. 

The  dead  who  die  of  infectious  diseases  should  be 
washed  in  a  strong  disinfectant  solution,  and  should  be 
given  a  private  funeral  in  which  the  body,  if  exposed, 
should  not  be  touched.  In  my  judgment,  the  body 
is  best  disposed  of  by  cremation. 


CHAPTER    VI. 
CULTIVATION  OF  BACTERIA;    CULTURE-MEDIA. 

ACCURACY  of  observation  requires  that  the  bacteria  be 
separated  from  their  natural  surroundings  and  artificially 
grown  upon  certain  prepared  media  of  standard  compo- 
sition, in  such  a  manner  that  only  organisms  of  the  same 
kind  are  together. 

One  after  another  various  organic  and  inorganic  mix- 
tures have  been  suggested,  but,  although  almost  any 
compound  containing  organic  matter,  even  in  small 
amounts,  will  suffice  for  the  nourishment  of  bacteria, 
a  certain  few  have  met  with  particular  favor  as  being 
most  valuable. 

Rather  than  give  a  complete  review  of  the  work  wjiich 
has  already  been  done,  in  the  following  pages  the  most 
useful  preparations  only  will  be  considered. 

Our  knowledge  of  the  biology  of  the  bacteria  has 
shown  that  they  grow  best  in  a  mixture  containing  at 
least  80  per  cent,  of  water,  of  a  neutral  or  feebly  alka- 
line reaction,  and  of  a  composition  which,  for  the  patho- 
genic forms  at  least,  should  approximate  the  juices  of 
the  animal  body.  It  might  be  added  that  transparency 
is  a  very  desirable  quality,  and  that  the  most  gener- 
ally useful  culture-media  are  those  that  can  be  readily 
liquefied  and  solidified. 

Bouillon  is  one  of  the  most  useful  and  most  simple  of 
the  media.  Its  preparation  is  as  follows  :  To  500  grams 
of  finely-chopped  lean,  boneless  beef,  1000  c.cm.  of  clean 
water  are  added  and  allowed  to  stand  for  about  twelve 
hours  on  ice.  At  the  end  of  this  time  the  liquor  is  de- 
canted, that  remaining  on  the  meat  expressed  through  a 
cloth,  and  then,  as  the  entire  quantity  is  seldom  regained, 

106 


CULTIVATION  OF  BACTERIA.  107 

enough  water  added  to  bring  the  total  amount  up  to  1000 
c.cm.  This  liquid  is  called  the  meat-infusion.  To  it 
10  grams  of  Whitte's  dried  beef-peptone  and  5  grams  of 
sodium  chlorid  are  added,  and  the  whole  boiled  until  the 
albumins  coagulate.  The  reaction  is  then  carefully  tested, 
in  order  that  whatever  sarcolactic  acid  may  have  been 
present  in  the  meat  may  be  neutralized  by  the  addition 
of  a  few  drops  of  a  saturated  aqueous  solution  of  sodium 
carbonate.  The  solution  is  added  drop  by  drop,  and  the 
reaction  frequently  tested  with  litmus-paper.  When  a 
neutral  reaction,  or,  better,  a  faint  alkaline  reaction,  is 
attained,  the  mixture  is  well  stirred,  boiled  again  for 
about  half  an  hour,  to  precipitate  the  alkaline  albumins 
formed,  and  filtered.  The  bouillon  thus  prepared  is  a 
clear  fluid  of  a  straw  color,  much  resembling  normal 
urine  in  appearance.  It  is  dispensed  in  tubes — about  10 
c.cm.  to  each — and  is  then  sterilized  by  steam  three  suc- 
cessive days  for  fifteen  to  twenty  minutes  each,  according 
to  the  directions  already  given  for  fractional  sterilization. 
(See  p.  94.) 

For  the  preparation  of  bouillon,  as  well  as  gelatin, 
agar-agar,  and  glycerin  agar  still  to  be  described,  beef- 
extract  (Liebig's)  may  be  employed,  but  for  the  most 
delicate  work  this  is  rather  undesirable,  because  of  its 
unstable  composition  and  because  of  the  precipitation  of 
meat-salts,  which  can  scarcely  be  filtered  out  of  the  agar- 
agar,  owing  to  the  fact  that  they  only  crystallize  when 
the  solution  cools.  When  it  is  desirable  to  prepare  the 
bouillon  from  beef-extract,  the  method  is  very  simple. 
To  1000  c.cm.  of  clean  water  10  grams  of  Whitte's  dried 
beef-peptone,  5  grams  of  sodium  chlorid,  and  about  2 
grams  of  beef-extract  are  added.  The  solution  is  boiled 
until  the  constituents  are  dissolved,  neutralized,  if  neces- 
sary, and  filtered  when  cold.  If  it  is  filtered  while  hot, 
there  is  always  a  subsequent  precipitation  of  meat-salts, 
which  clouds  it. 

Bouillon  and  other  liquid  culture-media  are  best  dis- 
pensed and  kept  in  small  receptacles — test-tubes  or  flasks 


io8 


PATHOGENIC  BACTERIA. 


— in  order  that  a  single  contaminating  organism,  should 
it  enter,  may  not  spoil  the  entire  bulk.  A  very  con- 
venient simple  apparatus  used  by  bacteriologists  for  fill- 
ing tubes  with  liquid  media  is  shown  in  Figure  17.  It 


FIG.  17. — Funnel  for  filling  tubes  with  culture-media  (Warren). 

need  not  be  sterilized  before  using,  as  the  culture-medium 
will  be  sterilized  by  the  intermittent  method  after  the 
tubes  are  filled.  The  test-tubes  and  flasks  into  which 
the  culture-medium  is  filled  must,  however,  be  previously 
sterilized  by  dry  heat.  The  dry-heat  sterilization  is  done, 
of  course,  after  the  cotton  plugs  are  in  place. 

Bouillon  is  the  basis  of  most  of  the  culture-media. 
The  addition  of  10  per  cent,  of  gelatin  makes  it  "  gela- 
tin ;"  that  of  i  per  cent,  of  agar-agar  makes  it  "agar- 
agar. ' '  The  preparation  of  these  media,  however,  varies 
somewhat  from  that  of  the  plain  bouillon. 


CULTIVATION  OF  BACTERIA.  109 

Gelatin. — The  culture-medium  known  as  gelatin  has  de- 
cided advantages  over  the  bouillon,  not  only  because  it  is 
an  excellent  food  for  bacteria,  and,  like  the  bouillon,  trans- 
parent, but  because  it  is  also  solid.  Nor  is  this  all  :  it  is 
a  transparent  solid  which  can  be  made  liquid  or  solid  at 
will.  It  is  prepared  as  follows  :  To  1000  c.  cm.  of  meat- 
infusion  or  to  1000  c.cm.  of  water  containing  2  grams  of 
beef-extract  in  solution,  10  grams  of  peptone,  5  grams  of 
salt,  and  100  grams  of  gelatin  ("Gold  label "  is  the  best 
commercial  article)  are  added,  and  boiled  for  about  an 
hour  over  a  moderately  hot  flame.  Double  boilers  are 
very  slow,  and  if  proper  care  is  exercised  there  is  little 
danger  of  the  gelatin  burning.  It  must  be  stirred  occa- 
sionally, and  the  flame  should  be  so  distributed  by  wire 
gauze  as  not  to  act  upon  a  single  point  of  the  bottom  of 
the  kettle.  At  the  end  of  the  hour  the  albumins  of 
the  meat-infusion  will  be  coagulated  and  the  gelatin 
thoroughly  dissolved.  Gunther  has  shown  that  the 
gelatin  congeals  better  if  allowed  to  dissolve  slowly  in 
warm  water  before  boiling.  The  liquid  is  now  cooled 
to  60°  C.  and  neutralized — i.  e.  alkalinized.  As  the  gela- 
tin is  itself  acid,  a  relatively  larger  amount  of  the  sodium- 
carbonate  solution  will  be  needed  than  was  required  for 
the  bouillon.  When  the  proper  reaction  is  attained,  as 
much  water  as  has  been  lost  by  vaporization  during  the 
process  of  boiling,  intimately  mixed  with  the  white  of 
an  egg,  is  added,  well  stirred  in,  and  the  whole  boiled 
for  half  an  hour,  then  filtered. 

If  the  filter-paper  be  of  good  quality  and  properly 
folded  (pharmaceutical  filter),  and  if  the  gelatin  be  prop- 
erly dissolved,  the  whole  quantity  should  pass  through 
before  cooling  too  much.  Should  only  half  go  through 
before  cooling,  the  remainder  must  be  returned  to  the 
pot,  heated  to  boiling  once  more,  and  then  passed  through 
a  new  filter-paper.  As  a  matter  of  fact,  gelatin  generally 
filters  readily.  A  wise  precaution  is  to  catch  the  first  few 
centimeters  in  a  test-tube  and  boil  them,  so  that  if  a 
cloudiness  shows  the  presence  of  uncoagulated  albumin, 


no  PATHOGENIC  BACTERIA. 

the  whole  mass  can  be  boiled  again.  The  finished  gel- 
atin is  at  once  distributed  into  sterilized  tubes,  and  then 
sterilized  like  the  bouillon  by  the  fractional  method. 

Of  course,  the  gelatin  or  any  other  culture-medium  can 
be  kept  en  masse  indefinitely,  but  should  a  contaminating 
micro-organism  accidentally  enter,  the  whole  quantity 
will  be  spoiled  ;  if,  on  the  other  hand,  it  is  kept  in  tubes, 
several  of  them  may  be  lost  without  much  inconvenience. 
Under  proper  precautions  of  sterilization  and  protection 
it  should  all  keep  well. 

Agar-agar. — Agar-agar  is  the  commercial  name  of  a 
Japanese  sea- weed  which  dissolves  in  boiling  water  with 
resulting  thick  jelly  when  cold.  The  jelly,  which  solidi- 
fies between  30°  and  40°  C.,  cannot  again  be  melted  ex- 
cept by  the  elevation  of  its  temperature  to  the  boiling- 
point,  so  that  this  culture-medium,  which  is  nearly  trans- 
parent, is  almost  as  useful  as  gelatin.  In  addition  to  its 
readiness  to  liquefy  and  solidify,  it  is  sufficiently  firm 
to  allow  of  the  incubation-temperature — i.  e.  37°  C. — at 
which  gelatin  is  always  liquid,  and  no  better  than  bouillon. 

The  preparation  of  this  medium  is  generally  described 
in  the  text-books  as  one  u  requiring  considerable  patience 
and  much  waste  of  filter- paper. "  In  reality,  it  is  not  dif- 
ficult if  a  good  heavy  filter-paper  be  obtained  and  no 
attempt  be  made  to  filter  the  solution  until  the  agar-agar 
is  perfectly  dissolved.  It  is  prepared  as  follows  :  To  1000 
c.cm.  of  bouillon  made  as  described  above,  preferably  of 
meat  instead  of  beef-extract,  10  grams  of  agar-agar  are 
added.  The  mixture  is  boiled  for  an  hour,  or,  if  possible, 
two.  At  the  end  of  the  first  hour  it  is  cooled  to  about 
60°  C.,  and  after  neutralization,  which  may  not  be  neces- 
sary if  the  bouillon  was  neutral,  an  egg  beaten  up  in 
water  is  added,  and  the  liquid  is  boiled  again  until  the 
egg  is  entirely  coagulated.  The  reaction  of  the  agar-agar 
should  be  neutral  rather  than  alkaline,  as,  for  an  un- 
known reason,  alkalinity  seems  to  interfere  slightly  with 
filtration. 

After  the  boiling,  which  should  be  brisk,  has  caused 


CUL 77 VA TION  OF  BA CTERIA .  in 

the  thorough  solution  of  the  agar-agar,  it  is  filtered,  just 
as  the  gelatin  was,  through  a  carefully-folded  pharmaceu- 
tical filter  wet  with  boiling  water.  It  may  expedite  mat- 
ters to  pour  in  about  one-half  of  the  solution,  keep  the 
remainder  hot,  and  subsequently  add  it  when  necessary. 
Experience  shows  that  1000  c.cm.  of  agar-agar  rarely  go 
through  one  paper,  and  I  always  expect  when  beginning 
the  filtration  to  be  compelled  to  boil  the  material  which 
remains  on  the  paper  again,  and  pour  it  through  a  new 
filter. 

The  formerly  much-employed  hot-water  and  gas-jet 
filters  seem  unnecessary.  If  properly  prepared,  the  whole 
quantity  will  filter  in  from  fifteen  to  thirty  minutes. 

If  made  from  beef-extract,  the  agar-agar  almost  always 
precipitates  a  considerable  amount  of  meat-salts  as  it 
cools.  This  should  be  anticipated,  but,  so  far  as  I  can 
determine,  cannot  always  be  prevented.  The  amount  is 
certainly  lessened  by  making  the  bouillon  first,  filtering 
it  cold,  then  adding  the  agar-agar,  and  dissolving  and 
filtering  it. 

The  difficulty  of  filtering  the  agar-agar  has  led  Fliigge 
and  others  to  adopt  a  method  of  sedimentation.  An  in- 
genious apparatus  for  this  purpose  has  lately  been  devised 
by  Bleisch.  The  methods  can  be  simplified  by  using  a 
small  pharmaceutical  percolator,  the  bottom  of  which  is 
closed  by  a  rubber  cork  containing  a  tube  which  extends 
nearly  to  the  top  of  the  percolator  and  is  attached  to 
a  rubber  tube  with  a  pinchcock  below.  The  melted  agar- 
agar  is  poured  into  this,  and  kept  in  the  steam  apparatus 
until  the  sedimentation  is  sufficient  to  allow  clear  fluid  to 
be  drawn  from  the  top.  As  the  clear  agar-agar  is  drawn 
off  the  tube  is  pulled  down  through  the  rubber  cork,  and 
more  drawn  off  until  only  the  sediment  is  left. 

Agar-agar  is  dispensed  in  tubes  like  the  gelatin  and 
bouillon,  sterilized  by  steam  by  the  intermittent  process, 
and  after  the  last  sterilization,  before  cooling,  each  tube 
is  inclined  against  a  slight  elevation,  so  as  to  offer  an  ex- 
tensive flat  surface  for  the  culture. 


112  PATHOGENIC  BACTERIA. 

After  the  agar-agar  jelly  solidifies  its  contraction  causes 
some  water  to  collect  at  the  lower  part  of  the  tube.  This 
should  not  be  removed,  as  it  keeps  the  material  moist, 
and  also  because  it  has  a  distinct  influence  upon  the  cha- 
racter of  the  growth  of  the  bacteria. 

Glycerin  Agar-agar. — For  an  unknown  reason  certain 
of  the  bacteria  which  will  not  grow  upon  the  agar-agar 
as  prepared  above  will  do  so  if  3-7  per  cent,  of  glycerin 
be  added.  Among  these  is  the  tubercle  bacillus,  which, 
not  growing  at  all  upon  plain  agar-agar,  will  grow  well 
when  glycerin  is  added — a  fact  discovered  by  Roux  and 
Nocard.  The  glycerin  may  also  be  added  to  gelatin  or 
any  other  medium. 

Blood-serum. — The  great  advantage  possessed  by  this 
medium  is  that  it  is  itself  a  constituent  of  the  body,  and 
hence  offers  opportunities  for  the  development  of  the 
parasitic  forms  of  bacteria  under  the  most  natural  con- 
ditions possible.  It  is  the  most  difficult  of  all  the  media 
to  prepare.  The  blood  must  be  obtained  from  a  slaughter- 
house in  an  appropriate  receptacle,  the  best  things  for  the 
purpose  being  tall  narrow  jars  of  about  i  liter  capacity, 
with  a  tightly-fitting  lid.  The  jars  are  sterilized  by  heat 
or  by  washing  with  alcohol  and  ether,  are  carefully  dried, 
closed,  and  carried  to  the  slaughter-house  where  the  blood 
is  to  be  obtained.  As  the  blood  flows  from  the  severed 
vessels  of  the  animal  the  jars  are  filled  one  by  one.  It 
seems  advisable  to  allow  the  first  blood  to  escape,  as  it  is 
likely  to  become  contaminated  from  the  hair.  By  waiting 
until  a  coagulum  forms  upon  the  hair  the  danger  of  con- 
tamination is  obviated.  The  jars  when  full  are  allowed 
to  stand  undisturbed  until  quite  firm  coagula  form  within 
them.  If  these  have  any  tendency  to  cling  to  the  glass, 
each  one  should  be  given  a  few  violent  twists,  so  as  to 
break  away  the  fibrinous  attachments.  After  this  the 
jars  are  carried  to  the  laboratory  and  stood  upon  ice  for 
forty-eight  hours,  by  which  time  the  clots  will  have  re- 
tracted considerably,  and  a  moderate  amount  of  clear 
serum  can  be  removed  by  sterile  pipettes  and  placed  in 


CULTIVATION  OF  BACTERIA.  113 

sterile  tubes.  If  the  serum  obtained  is  red  and  clouded 
from  the  presence  of  corpuscles,  it  may  be  pipetted  into 
sterile  cylinders  and  allowed  to  sediment  for  twelve  hours, 
then  repipetted  into  tubes.  It  is  evident  that  such  com- 
plicated maneuvring  will  offer  many  possible  chances  of 
infection  ;  hence  the  sterilization  of  the  serum  is  of  the 
greatest  importance. 

If  it  is  desirable  to  use  the  serum  as  a  liquid  medium,  it 
is  exposed  to  a  temperature  of  60°  to  65°  C.  for  one  hour 
upon  each  of  five  consecutive  days.  If  it  is  thought  best 
to  coagulate  the  serum  and  make  a  solid  culture-medium, 
it  may  be  exposed  twice,  for  an  hour  each  time — or  three 
times  if  there  is  distinct  reason  to  think  it  contam- 
inated— to  a  temperature  just  short  of  the  boiling-point. 
During  the  process  of  coagulation  the  tubes  should  be 
inclined,  so  as  to  offer  a  large  surface  for  the  growth  of 
the  culture.  The  serum  thus  prepared  may  be  white,  or 
have  a  reddish-gray  color  if  many  corpuscles  are  pres- 
ent, and  is  opaque.  It  cannot  be  melted,  but  once  solid 
remains  so. 

Koch  devised  a  very  good  apparatus  (Fig.  18)  for  coag- 


FIG.  18. — Koch's  apparatus  for  coagulating  and  sterilizing  blood-serum. 

ulating   blood-serum.     The   bottom   should   be   covered 
with  cotton,  a  single  layer  of  tubes  placed  upon  it,  and 


H4  PA  THOGENIC  BA  CTERIA . 

the  temperature  elevated  until  coagulation  occurs.  The 
repeated  sterilizations  may  be  conducted  in  this  apparatus, 
or  may  be  done  equally  well  in  the  steam  apparatus,  the 
cover  of  which  is  not  completely  closed,  for  if  the  tem- 
perature of  the  serum  is  raised  too  high  it  is  certain  to 
bubble. 

Loffler's  blood-serum  mixture,  which  seems  rather 
better  for  the  cultivation  of  some  species  than  the  blood- 
serum  itself,  consists  of  i  part  of  a  beef-infusion  bouillon 
containing  i  per  cent,  of  glucose  and  3  parts  of  liquid 
blood-serum.  After  being  well  mixed  this  is  distributed 
in  tubes,  and  sterilized  and  coagulated  like  the  blood- 
serum  itself.  Most  organisms  grow  more  luxuriantly 
upon  it  than  upon  either  plain  blood-serum  or  other 
culture-media.  Its  special  usefulness  is  for  the  Bacillus 
diphtheriae,  which  grows  upon  it  with  rapidity  and  with 
quite  a  characteristic  appearance. 

Potatoes. — Without  taking  time  to  review  the  old 
method  of  boiling  potatoes,  opening  them  with  sterile 
knives,  and  protecting  them  in  the  moist  chamber,  or 
the  much  more  easily  conducted  method  of  Ksmarch  in 
which  the  slices  of  potato  are  sterilized  in  the  small 
dishes  in  which  they  are  afterward  kept  and  used,  we 
will  at  once  pass  to  what  seems  the  most  simple  and 
satisfactory  method  of  using  this  valuable  medium — that 
of  Bolton  and  Globig  : 

With  the  aid  of  a  cork-borer  a  little  smaller  in  diam- 
eter than  the  test-tube  ordinarily  used  a  number  of  cyl- 
inders are  cut  from  potatoes.  Rather  large  potatoes 
should  be  used,  the  cylinders  being  cut  transversely,  so 
that  a  number,  each  about  an  inch  and  a  half  in  length, 
can  be  cut  from  one  potato.  The  skin  is  removed  from 
the  cylinders  by  cutting  off  the  ends,  after  which  each 
cylinder  is  cut  in  two  by  an  oblique  incision,  so  as  to 
leave  a  broad,  flat  surface.  The  half-cylinders  are  placed 
each  in  a  test-tube  previously  sterilized,  and  then  are 
exposed  three  times,  for  half  an  hour  each,  to  the  pass- 
ing steam  of  the  sterilizer.  This  steaming  cooks  the 


CULTIVATION  OF  BACTERIA.  115 

potato  and  also  sterilizes  it.  Such  cultures  are  apt  to 
deteriorate  rapidly,  first  by  turning  very  dark ;  second, 
by  drying  so  as  to  be  useless.  Abbott  has  shown  that 
if  the  cut  cylinders  be  allowed  to  stand  for  twelve  hours 
in  running  water  before  being  dispensed  in  the  tubes, 
they  do  not  turn  dark.  Drying  may  be  prevented  by 
adding  a  few  drops  of  clean  water  to  each  tube  before 
sterilizing.  It  is  not  necessary  to  have  a  special  small 
chamber  blown  in  the  tube  to  contain  this  water ;  only 
a  small  quantity  need  be  added,  and  this  will  not  touch 
the  potato,  which  does  not  reach  the  bottom  of  the 
rounded  tube. 

A  potato-juice  has  also  been  suggested,  and  is  of  some 
value.  It  is  made  thus  :  To  300  c.cm.  of  water  100  grams 
of  grated  potato  are  added,  and  allowed  to  stand  on  ice 
over  night.  Of  the  pulp  300  c.cm.  are  expressed  through 
a  cloth  and  cooked  for  an  hour  on  a  water-bath.  After 
cooking,  the  liquid  is  filtered  and  receives  4  per  cent,  of 
glycerin.  It  may  or  may  not  need  neutralization.  Upon 
this  medium  the  tubercle  bacillus  grows  well,  especially 
when  the  reaction  of  the  medium  is  acid,  but  loses  its 
virulence. 

Milk. — Milk  is  useful  as  a  culture-medium.  As  when 
the  milk  stands  the  cream  which  rises  to  the  top  is  a 
source  of  inconvenience,  it  is  best  to  secure  from  a  dairy 
fresh  milk  from  which  the  cream  has  been  removed  by 
a  centrifugal  machine.  It  is  placed  in  sterile  tubes  and 
sterilized  by  steam  by  the  intermittent  method.  The 
opaque  nature  of  this  culture-medium  often  permits  the 
undetected  development  of  contaminating  organisms. 
A  careful  watch  should  therefore  be  kept  upon  it  lest  it 
spoil. 

Litmus  Milk. — This  is  milk  to  which  just  enough  of 
a  saturated  watery  solution  of  pulverized  litmus  is  added 
to  give  a  distinct  blue  color.  Cow's  milk  is  inclined  to 
be  acid  in  reaction,  and  a  small  amount  of  sodium  car- 
bonate may  be  necessary  to  give  it  a  distinct  blue.  The 
use  of  litmus  is  probably  the  best  method  of  determining 


1 16  PA  THOGENIC  BACTERIA . 

whether  bacteria  by  their  growth  produce  acids  or  alka- 
lies. 

The  watery  solution  of  litmus,  being  a  vegetable  in- 
fusion, is  likely  to  spoil,  hence  should  always  be  treated 
like  the  culture-media  and  sterilized  by  steam  every  time 
the  receptacle  in  which  it  is  kept  is  opened. 

Peptone  solution,  or  Dunham's  solution,  is  very  use- 
ful for  the  detection  of  certain  faint  colors.  It  is  a  per- 
fectly clear,  colorless  solution,  made  as  follows : 

Sodium  chlorid,  0.5  ^  Boil  until  the  ingredients 

Whitte's  dried  peptone,   i.      >    dissolve  ;  then  filter,  fill 
Water,  100.     J     into  tubes,  and  sterilize. 

It  is  the  best  medium  for  the  detection  of  indol.  In  it 
the  Bacillus  pyocyaneus  produces  its  blue  color.  The 
addition  of  4  c.cm.  of  the  following  solution — 

Rosalie  acid,  0.5, 

80  per  cent,  alcohol,  100. 

makes  it  become  an  excellent  reagent  for  the  detection 
of  acids  and  alkalies.  The  solution  is  pale  rose  in  color. 
If  the  bacterium  produces  acids,  the  color  fades  ;  if  alka- 
lies, it  intensifies. 

It  is  not  intended  that  the  student  shall  infer  that 
there  are  no  culture-media  other  than  these,  which  have 
been  selected  because  of  their  iisefulness  and  popularity. 
Many  other  compounds  and  as  many  simple  substances 
are  employed  ;  for  example,  eggs,  white  of  egg,  urine, 
bread,  sputum,  sugar  solutions,  hydrocele  fluid,  and 
aqueous  humor. 


CHAPTER    VII. 
CULTURES,  AND  THEIR  STUDY. 

THE  objects  which  we  have  had  before  us  in  the  prep- 
aration of  the  culture-media  were  numerous.  We  have 
prepared  them  so  as  to  allow  us  to  separate — or,  rather, 
to  isolate — bacteria,  to  keep  them  in  healthy  growth  for 
considerable  lengths  of  time,  to  enable  us  to  observe  their 
biologic  peculiarities,  and  to  introduce  them  without  dif- 
ficulty into  the  bodies  of  animals. 

The  isolation  of  bacteria  was  impossible  until  the  fluid 
culture-media  of  the  early  observers  were  replaced  by  the 
solid  media,  and  was  exceedingly  crude  until  Koch  gave 
us  the  solid,  transparent  media  and  the  well-known 
u  plate-cultures. " 

A  growth  of  artificially-planted  micro-organisms  in 
which  an  immense  number  are  massed  together  is  called 
a  culture.  If  such  a  growth  contains  but  one  kind  of 
organism,  it  is  known  as  a  ptire  culture. 

It  has  become  the  habit  at  present  to  use  the  term  "cul- 
ture" rather  loosely,  so  that  it  does  not  always  signify  a 
growth  of  micro-organisms  artificially  planted,  but  may 
signify  a  growth  taking  place  under  natural  conditions ; 
thus,  typhoid  bacilli  are  said  to  exist  in  the  spleens  of 
patients  dead  of  that  disease  u  in  pure  culture,"  because 
no  other  bacteria  are  there  ;  and  sometimes,  when  in  ex- 
pectorated fragments  of  cheesy  matter  from  tuberculosis 
pulmonalis  the  tubercle  bacilli  are  very  numerous  and 
unmixed  with  other  bacteria,  the  term  upure  culture" 
is  again  used  to  describe  the  condition. 

Three  principal  methods  are  at  present  employed  to 
enable  us  to  secure  pure  cultures  of  bacteria,  but  before 
beginning  a  description  of  them  it  is  well  to  observe  that 

117 


n8 


PATHOGENIC  BACTERIA. 


the  peculiarities  of  certain  pathogenic  forms  enable  us 
to  use  special  means,  taking  advantage  of  their  eccentrici- 
ties, for  their  isolation,  and  that  the  general  methods  are 
in  reality  more  useful  for  the  non-pathogenic  than  for  the 
pathogenic  forms. 

All  three  methods  depend  upon  the  observation  of 
Koch,  that  when  germs  are  equally  distributed  through- 
out some  liquefied  nutrient  medium  which  can  be  solidi- 
fied in  a  thin  layer,  the  growth  of  the  germs  takes  place 
in  little  scattered  groups  or  families,  called  colonies,  dis- 
tinctly separated  from  each  other  and  capable  of.  trans- 
plantation to  tubes  of  culture-media. 

Plate-cultures. — The  plate-cultures,  originally  made 
by  Koch,  require  considerable  apparatus,  and  of  late  years 
have  given  place  to  the  more  ready  methods  of  Petri  and 
Von  Esmarch.  So  great,  however,  is  the  historic  interest 
attached  to  the  plates  that  it  would  be  a  great  omission 
not  to  describe  Koch's  method  in  full. 

Apparatus. — Half  a  dozen  glass  plates,  about  6  by  4 
inches  in  size,  free  from  bubbles  and  scratches  and 
ground  at  the  edges,  are  carefully  cleaned,  placed  in  a 
sheet-iron  box  made  to  receive  them,  and  then  put  in 

the  hot-air  closet,  where 
they  are  sterilized.  The  box, 
which  is  tightly  closed,  al- 
lows the  sterilized  plates  to 
be  kept  on  hand  indefinitely 
before  using. 

A  moist  chamber,  or  double 
dish,  about  10  inches  in  di- 
ameter and  3  inches  deep,  the 
upper  half  being  just  enough 
larger  than  the  lower  to  allow 
it  to  close  over  it,  is  carefully 
washed.  A  sheet  of  bibulous 
paper  is  placed  in  the  bottom,  so  that  some  moisture  can 
be  retained,  and  a  i  :  1000  bichlorid  solution  is  poured  in 
and  brought  in  contact  with  the  sides,  top,  and  bottom 


FIG.  19. — Complete  levelling  appa- 
ratus for  pouring  plate-cultures,  as 
taught  by  Koch. 


CULTURES,   AND    THEIR  STUDY.  119 

by  turning  the  dish  in  all  directions.  The  solution  is 
emptied  out,  and  the  dish,  which  is  always  kept  closed, 
is  ready  for  use. 

A  levelling  apparatus  is  required  (Fig.  19).  This  con- 
sists of  a  wooden  tripod  with  adjustable  screws,  and  a  glass 
dish  covered  by  a  flat  plate  of  glass  upon  which  a  low 
bell-jar  stands.  The  glass  dish  is  filled  with  broken  ice 
and  water,  covered  with  the  glass  plate,  and  then  exactly 
levelled  by  adjusting  the  screws  under  the  legs  of  the 
tripod.  When  level  the  cover  is  placed  upon  it,  and  it 
is  ready  for  use. 

Method  (Fig.  20). — A  sterile  platinum  loop  is  dipped 
into  the  material  to  be  examined,  a  small  quantity  se- 


FIG.  20. — Method  of  holding  tubes  during  inoculation. 

cured,  and  stirred  about  so  as  to  distribute  it  evenly 
through  a  tube  of  the  melted  gelatin.  If  the  material 
under  examination  is  very  rich  in  bacteria,  one  loopful 
may  contain  a  million  individuals,  which,  if  spread  out 
in  a  thin  layer,  would  develop  so  many  colonies  that  it 
would  be  impossible  to  see  any  one  clearly ;  hence  the 
necessity  for  a  dilution.  From  the  first  tube  a  loopful 
of  gelatin  is  carried  to  a  second  tube  of  melted  gelatin 
and  stirred  well,  so  as  to  distribute  the  organisms  evenly 
through  it.  In  this  tube-  we  may  have  no  more  than  ten 
thousand  organisms,  and  if  the  same  method  of  dilution 
be  used  again,  the  third  tube  may  have  only  a  few  hun- 
dreds, and  a  fourth  only  a  few  dozen  colonies. 

After  the  tubes  are  prepared,  one  of  the  sterile  glass 
plates  is  caught  by  its  edges,  removed  from  the  iron  box, 
and  placed  beneath  the  bell-glass  upon  the  cold  plate 


120  PA  THOGENIC  BA  CTERIA . 

covering  the  ice-water  of  the  levelling  apparatus.  The 
plug  of  cotton  closing  the  mouth  of  tube  No.  i  is  re- 
moved, and  to  prevent  contamination  during  the  outflow 
of  the  gelatin  the  mouth  of  the  tube  is  held  in  the  flame 
of  a  Bunsen  burner  for  a  moment  or  two.  The  gelatin 
is  then  cautiously  poured  out  upon  the  plate,  the  mouth 
of  the  tube,  as  well  as  the  plate,  being  covered  by  the 
bell-glass  to  prevent  contamination  by  germs  in  the  air. 
The  apparatus  being  level,  the  gelatin  spreads  out  in  an 
even,  thin  layer,  and,  the  plate  being  cold  from  the  ice 

beneath,  it  immediately  solidi- 
fies, and  in  a  few  moments  can 
be  removed  to  the  moist  cham- 
ber prepared  to  receive  it.  As 

FIG.  21.— Glass  bench. 

soon  as  plate  No.  i  is  prepared, 

the  contents  of  tube  No.  2  are  poured  upon  plate  No.  2, 
allowed  to  spread  out  and  solidify,  and  then  superimposed 
on  plate  No.  i  in  the  moist  chamber,  being  separated  from 
the  plate  already  in  the  chamber  by  small  glass  benches 
(Fig.  21)  made  for  the  purpose  and  sterilized.  After  the 
contents  of  all  the  tubes  are  thus  distributed,  the  moist 
chamber  and  its  contents  are  allowed  to  stand  for  some 
hours,  to  permit  the  bacteria  to  grow.  Where  each  or- 
ganism falls  a  colony  develops,  and  the  success  of  the 
whole  method  depends  upon  the  isolation  of  a  colony 
and  its  transfer  to  a  tube  of  culture-medium  where  it 
can  grow  unmixed  and  undisturbed. 

The  description  must  have  made  evident  the  fact  that 
only  such  culture-media  can  be  used  for  plate-cultures  as 
can  be  melted  and  solidified  at  will — viz.  gelatin,  agar- 
agar,  and  glycerin  agar-agar.  Blood-serum  and  Loffler's 
mixture  are  entirely  inappropriate. 

The  great  drawback  to  this  excellent  method  is  the 
cumbersome  apparatus  required  and  the  comparative  im- 
possibility of  making  plate-cultures,  as  is  often  desirable, 
in  the  clinic,  at  the  bedside,  or  elsewhere  than  in  the 
laboratory.  The  method  therefore  soon  underwent  mod- 
ifications, the  most  important  being 


CULTURES,   AND    THEIR  STUDY.  121 

Petri's  Dishes. — These  small  dishes  (Fig.  22),  about 
4  inches  in  diameter  and  y2  inch  deep,  with  accurately 
fitting  lids,  are  about  as  convenient  as  anything  that  has 
been  devised  in  bacteriological  technique.  They  dis- 


FIG.  22. — Petri  dish  for  making  plate-cultures. 

pense  with  plates  and  plate-boxes,  with  moist  chambers 
and  benches,  and  usually  with  the  levelling  apparatus, 
though  this  is  still  employed  in  connection  with  the 
Petri  dishes  in  some  laboratories. 

The  method  of  the  employment  of  Petri  dishes  is  very 
simple.  The  dishes  are  carefully  cleaned,  polished,  and 
sterilized  by  hot  air,  care  being  taken  that  they  are  placed 
in  the  hot-air  closet  right  side  up,  and  after  sterilization 
are  kept  covered  and  in  that  position.  The  dilution  of 
the  material  under  examination  is  made  with  gelatin  or 
agar-agar  tubes  in  the  manner  described  above,  the  plugs 
are  removed,  the  mouth  of  the  tube  is  cautiously  held 
for  a  moment  in  the  flame,  then  the  contents  of  each 
tube  are  poured  into  one  of  the  sterile  dishes,  whose  top 
is  elevated  just  sufficiently  to  allow  the  mouth  of  the 
tube  to  enter.  The  gelatin  is  spread  over  the  bottom 
of  the  dish  in  an  even  layer,  is  allowed  to  solidify, 
labelled,  and  then  stood  away  for  the  colonies  to  develop. 

Esmarch  Tubes. — This  method,  devised  by  Esmarch, 
converts  the  walls  of  the  test-tube  into  the  plate  and  dis- 
penses with  all  other  apparatus.  The  tubes,  which  are 
inoculated  and  in  which  the  dilutions  are  made,  should 
contain  less  than  half  the  usual  amount  of  gelatin  or 
agar-agar.  After  inoculation  the  cotton  plugs  are  pushed 
into  the  tubes  until  even  with  their  mouths,  and  then 
covered  with  a  rubber  cap,  which  protects  them  from 
wetting.  A  groove  is  next  cut  in  a  block  of  ice,  and 


122  PATHOGENIC  BACTERIA. 

the  tube,  held  almost  horizontally,  is  rolled  in  this  until 
the  entire  surface  of  the  glass  is  covered  with  a  thin 
layer  of  the  solid  medium  (Fig.  23).  Thus  the  tube 
becomes  the  plate  upon  which  the  colonies  develop. 


FIG.  23. — Esmarch  tube  on  block  of  ice  (redrawn  after  Abbott). 

Several  little  points  need  to  be  observed  in  carrying 
out  Bsmarch's  method.  The  tube  must  not  contain  too 
much  culture-medium,  or  it  cannot  be  rolled  into  an  even 
layer.  In  rolling  the  contents  should  not  touch  the  cotton 
plug,  lest  it  be  glued  to  the  glass  and  its  subsequent  use- 
fulness be  injured.  No  water  must  be  admitted  from  the 
melted  ice. 

The  offspring  of  each  bacterium  growing  upon  the 
film  of  gelatin  constituting  a  plate-culture  form  a  mass 
which  has  already  been  pointed  out  as  a  colony.  These 
small  bacterial  families  may  be  seen  through  a  micro- 
scope when  still  much  too  small  for  detection  by  the 
naked  eye,  and  because  of  their  minuteness  should  always 
be  studied  with  the  microscope. 

The  original  plates  of  Koch  are  very  inconvenient  for 
such  examination,  because  it  is  impossible  to  remove 
them  from  the  moist  chamber  and  lay  them  upon  the 
stage  of  the  microscope  without  exposing  them  to  the 
danger  of  contamination  by  the  atmosphere,  so  that  the 
advantages  Nof  Petri  dishes  and  Bsmarch  tubes,  where 
the  examination  may  be  made  through  the  glass  tube  or 


CULTURES,   AND    THEIR  STUDY. 


123 


through  the  bottom  of  the  inverted  dish,  will  be  more 
than  ever  apparent. 

The  colonies  should  be  viewed  from  time  to  time  in 
their  growth,  drawings  being  made  of  the  appearances, 
so  as  to  form  a  series  showing  the  developmental  cycle. 
Most  colonies  will  be  found  to  originate  as  spherical,  cir- 
cumscribed, slightly  granular,  yellowish,  greenish,  or 
brownish  dots,  and  later  to  send  out  offshoots  or  filaments 
or  to  develop  concentric  rings  or  characteristic  liquefac- 
tions. A  few  appear  from  the  very  first  as  woolly  clumps 
of  entangled  threads. 

Some  of  the  most  diverse  forms  of  colonies  are  repre- 
sented in  the  accompanying  illustrations  (Figs.  24-28). 


FIGS.  24,  25,  26. — The  various  appearances  of  colonies  of  bacteria  under  the 
microscope :  a,  colony  of  Bacillus  liquefaciens  parvus  (Luderitz) ;  b,  colony 
of  Bacillus  polypiformis  (Liborius);  c,  colony  of  Bacillus  radiatus  (Luderitz). 

A  pure  culture,  when  obtained  from  colonies  growing 
upon  a  plate,  must  always  be  made  from  a  single  colony, 
the  transplantation  being  accomplished  under  a  low  power 
of  the  microscope.  The  naked  eye  can  rarely  be  depended 
upon  to  recognize  the  purity  of  a  colony  or  its  isolation. 

Selecting  as  isolated,  large,  and  characteristic  a  colony 
as  possible,  it  is  brought  to  the  centre  of  the  field.  A 
platinum  wire,  securely  fused  into  a  glass  handle  about 
8  inches  long,  is  sterilized  by  being  made  incandescent 
in  a  Bunsen  flame,  cooled,  and  then  cautiously  manipu- 
lated until,  while  it  is  watched  through  the  microscope, 


124 


PATHOGENIC  BACTERIA. 


it  is  seen  to  touch  the  colony  and  take  part  of  its  con- 
tents away.  In  this  maneuvre  the  wire  must  not  touch 
the  objective,  the  glass,  or  anything  except  the  colony* 
Having  secured  the  adhesion  of  a  few  bacteria  to  the 
sterile  wire,  the  pure  culture  is  made  by  introducing 
them  into  a  sterile  culture-medium. 

If  the  pure  culture  is  to  be  made  in  bouillon,  the  tube 
is  held  obliquely,  so  that  when  the  cotton  plug  is  cau- 
tiously removed  no  germs  can  fall. in  from  the  air.  The 
plug  is  removed  by  a  twisting  movement.  The  wire,  with- 
out being  allowed  to  touch  the  mouth  or  sides  of  the 
tube,  is  plunged  into  its 
contents  and  stirred  about 
until  the  bacteria  are  de- 
tached, and  is  then  re- 


FlGS.  27,  28. — The  various  appearances  of  colonies  of  bacteria  under  the 
microscope  :  a,  colony  of  Bacillus  muscoides  (Liborius) ;  b,  colony  of  Bacillus 
anthracis  (Fliigge). 

moved  and  the  plug  replaced.  The  wire  should  be  im- 
mediately sterilized  by  heating  to  incandescence,  lest  the 
bacteria  be  pathogenic  and  capable  of  doing  subsequent 
harm. 

If  the  culture  is  to  be  made  in  gelatin,  a  different 
method  is  employed.  The  tube  is  either  held  horizon- 
tally, or,  as  is  perhaps  better,  inverted  ;  the  cotton  plug: 


CULTURES,   AND    THEIR  STUDY.  125 

is  removed  cautiously  ;  the  wire  bearing  the  bacteria 
from  the  colony  is  introduced  until  its  point  enters  the 
centre  of  the  gelatin,  and  is  then  carefully  pushed  6n 
until  a  vertical  puncture  from  the  surface  to  the  bottom 
of  the  gelatin  is  made.  This  is  the  puncture-culture — 
u  stichcultur  "  of  the  Germans. 

If  the  bacteria  are  only  to  be  planted  upon  the  surface 
of  the  culture-medium,  the  wire  is  drawn  over  the  surface 
of  a  tube  of  obliquely  solidified  gelatin,  agar-agar,  blood- 
serum,  etc.  with  a  steady,  slow  movement,  so  as  to  scatter 
the  germs  along  its  path  and  cause  the  development  of 
the  bacteria  in  an  enormous  colony  or  mass  of  colonies 
in  a  line  following  the  longest  diameter  of  the  exposed 
surface  from  end  to  end.  This  is  the  stroke-culture — 
ustrichcultur." 

The  method  of  holding  the  tubes,  cotton  plugs,  and 
platinum  wire  during  the  process  of  inoculation  is  shown 
in  Figure  20. 

Sometimes  it  is  desirable  to  preserve  an  entire  colored 
colony  as  a  microscopic  specimen.  To  do  this  a  perfectly 
clean  cover-glass,  not  too  large  in  size,  is  momentarily 
warmed,  then  carefully  laid  upon  the  surface  of  the 
gelatin  or  agar-agar  containing  the  colonies.  Sufficient 
pressure  is  applied  to  the  surface  of  the  glass  to  exclude 
bubbles  underneath,  but  the  pressure  must  not  be  too 
great,  as  it  may  destroy  the  integrity  of  the  colony. 
The  cover  is  gently  raised  by  one  edge,  and  if  successful 
the  whole  colony  or  a  number  of  colonies,  as  the  case 
may  be,  will  be  found  adhering  to  it.  It  is  treated 
exactly  as  any  other  cover-glass  preparation,  is  dried, 
fixed,  stained,  and  mounted,  and  kept  as  a  permanent 
specimen.  It  is  called  an  adhesion  preparation — "  klatsch 
praparat. ' ' 

The  development  of  bacteria  in  liquids  is  of  less  in- 
terest than  that  upon  solid  media.  The  growth  generally 
manifests  itself  by  a  diffuse  turbidity.  Sometimes  flocculi 
float  in  the  otherwise  clear  medium.  Some  forms  grow 
most  rapidly  at  the  surface  of  the  liquid,  and  produce  a 


126 


PATHOGENIC  BACTERIA. 


distinct  membranous  pellicle  called  a  mycoderma.  In 
such  a  growth  multitudes  of  degenerated  bacteria  and 
laVge  numbers  of  spores  are  to  be  observed.  On  the 
other  hand,  it  occasionally  happens  that  the  growth 
occurs  chiefly  below  the  surface,  and  may  produce  gelat- 
inous masses  which  are  known  as  zooglea. 

In  gelatin  the  bacteria  exhibit  a  great  variety  of  ap- 
pearances, many  of  which  are  beautiful  and  interesting. 
Certain  bacteria,  as  the  tubercle  bacillus,  will  not  grow 
at  all  upon  gelatin.  Some  forms  which  are  rigidly  ae- 
robic will  only  grow  upon  or  near  the  surface  ;  others, 
anaerobic,  only  in  the  deeper  parts.  The  majority,  how- 
ever, grow  both  upon  the  surface  and  in  the  puncture 
made  by  the  wire.  Sometimes  the  consistence  of  the 
gelatin  is  unaltered  ;  sometimes  it  is  liquefied  throughout, 
sometimes  only  at  the  surface.  Sometimes  offshoots  ex- 
tend from  the  colonies  into  the  gelatin,  giving  the  culture 


FIG.  29. — Various  forms  of  gelatin  puncture-cultures :  a,  Bacillus  typhi  ab- 
dominalis;  b,  B.  anthracis;  c,  B.  mycoides;  d,  B.  mesentericus  vulgatus ; 
e,  B.  of  malignant  edema ;  ft  B.  radiatis. 


a  bristling  appearance.     Figure  29  will  serve  to  illustrate 
different  varieties  of.  gelatin  growth. 

The  growth  in  gelatin  is  generally  so  far  removed  from 
the  walls  of  the  tube  (a  central  puncture  nearly  always 


CULTURES,   AND    THEIR  STUDY.  127 

being  made  in  the  culture-medium,  in  order  that  the 
growth  be  symmetrical)  that  it  is  next  to  impossible  to 
make  a  microscopical  examination  of  it  with  any  power 
beyond  that  given  by  a  hand-lens. 

Much  attention  has  been  given  of  late  to  the  preparation 
of  microtome  sections  of  the  gelatin  growth.  To  accom- 
plish this  the  glass  is  warmed  sufficiently  to  allow  the 
gelatin  to  be  removed  and  placed  in  Miiller's  fluid  (bi- 
chromate of  potassium  2. -2. 5,  sulphate  of  sodium  i, 
water  100),  where  it  is  hardened.  When  quite  firm  it 
is  washed  in  water,  passed  through  alcohols  ascending 
in  strength  from  50  to  100  per  cent.,  imbedded  in  cel- 
loidin,  cut  wet,  and  stained  like  a  section  of  tissue. 

A  ready  method  of  doing  this  has  been  suggested  by 
Winkler,  who  bores  a  hole  in  a  block  of  paraffin  with 
the  smallest-size  cork-borer,  soaks  the  block  in  bichlorid 
solution  for  an  hour,  pours  liquid  gelatin  into  the  cavity, 
allows  it  to  solidify,  inoculates  it  by  the  customary  punc- 
ture of  the  platinum  wire,  allows  it  to  develop  sufficiently, 
and  when  ready  cuts  the  sections  under  alcohol,  subse- 
quently staining  them  with  much-diluted  carbol-fuchsin. 

Very  pretty  museum  specimens  of  plate-  and  puncture- 
cultures  in  gelatin  can  be  made  by  simultaneously  killing 
the  micro-organisms  and  permanently  fixing  the  gelatin 
with  formalin,  which  can  either  be  sprayed  upon  the 
gelatin  or  applied  in  dilute  solution.  As  gelatin  fixed 
in  formalin  cannot  subsequently  be  liquefied,  such  prep- 
arations will  last  indefinitely. 

The  growths  which  occur  upon  agar-agar  are  in  many 
ways  less  characteristic  than  those  in  gelatin,  but  as  this 
medium  does  not  liquefy  except  at  a  high  temperature 
(100°  C.),  it  has  that  great  advantage  over  gelatin.  The 
colorless  or  almost  colorless  condition  of  the  preparation 
also  aids  in  the  detection  of  such  chromogenesis  as  may 
be  the  result  of  the  micro-organismal  growth. 

Sometimes  the  growth  is  colored,  sometimes  not ;  some- 
times the  production  of  a  soluble  pigment  colors  the 
agar-agar  as  well  as  the  growth  ;  sometimes  the  growth 


128  PATHOGENIC  BACTERIA. 

is  one  color  and  the  agar-agar  another.  Sometimes  the 
growth  is  filamentous,  sometimes  a  smooth,  shining  band. 
Occasionally  the  bacterium  does  not  grow  upon  agar-agar 
unless  glycerin  be  added  (tubercle  bacillus) ;  sometimes 
it  will  not  grow  even  then  (gonococcus). 

Still  less  characteristic  are  the  growths  upon  potato. 
Most  bacteria  produce  rather  smooth,  shining,  irregu- 
larly-extending growths,  which  often  show  very  beauti- 
ful colors. 

In  milk  and  litmus  milk  one  must  observe  the  presence 
or  absence  of  acid-production,  the  coagulation  which  may 
or  may  not  accompany  it,  and  the  subsequent  gelatiniza- 
tion  or  digestion  of  the  coagulum. 

Blood-serum  is  liquefied  by  some  bacteria.  The  ma- 
jority of  organisms  are  not  very  characteristic  in  their 
development  upon  it.  Others,  as  the  bacillus  of  diph- 
theria, are,  however,  characterized  by  their  shape,  color, 
and  rapidity  of  development  at  given  temperatures. 

While  most  of  the  saprophytic  bacteria  will  grow  well 
at  the  ordinary  temperature  of  a  well-warmed  room,  the 
important  pathogenic  forms  require  to  be  kept  at  the 
temperature  of  the  body.  To  do  this  accurately  an  in- 
cubating oven  becomes  a  necessity.  Various  forms,  of 
wood  and  metal,  are  in  the  market,  the  one  shown  in  the 
illustration  (Fig.  30)  being  one  of  the  newest  and  best. 

It  scarcely  need  be  pointed  out  that  gelatin  cultures 
cannot  be  grown  in  the  incubating  oven,  as  the  medium 
will  not  remain  solid  at  temperatures  above  20-22°  C. 


CULTURES,  AND  THEIR  STUDY. 


129 


FIG.  30. — New  model  incubating  oven  with  electro-regulator. 


CHAPTER    VIII. 
THE  CULTIVATION  OF  ANAEROBIC  BACTERIA. 

THE  cultivation  of  micro-organisms  which  will  not 
grow  where  the  least  amount  of  oxygen  is  present  is 
always  attended  with  much  difficulty,  and  can  seldom  be 
accomplished  with  certainty.  Many  methods  have  been 
suggested,  but  not  one  can  be  described  as  satisfactory. 

Koch  originally  cultivated  anaerobic  bacteria  upon 
plates  by  covering  the  surface  of  the  soft  gelatin  with  a 
thin  film  of  mica  previously  sterilized  by  incandescence. 
Some  anaerobic  forms  will  grow  quite  well  by  such  a 
simple  exclusion  of  the  air,  but  the  strictly  anaerobic 
forms  will  not  develop  at  all. 

Hesse  originated  the  plan,  still  sometimes  followed,  of 
making  a  deep  puncture  in  recently  boiled  and  rapidly 
sterilized  gelatin  or  agar-agar,  then  covering  the  surface 
with  sterilized  oil,  through  which  no  oxygen  was  sup- 
posed to  penetrate  (Fig.  31). 

Iviborius  suggested  the  plan  of  having  a  tube  nearly 
full  of  gelatin  or  agar-agar,  boiling  it  just  before  inocu- 
lation, so  as  to  expand  and  drive  out  whatever  air  it 
might  contain,  making  the  inoculation  while  the  culture- 
medium  was  still  fluid,  cooling  rapidly  in  ice-water,  and 
sealing  up  the  tube  in  a  blowpipe  as  near  the  surface  of 
the  gelatin  as  possible. 

Ksmarch  used  a  regular  u  Ksmarch  tube,"  into  the 
central  cavity  of  which  melted  sterile  gelatin  was  poured 
to  exclude  the  air. 

Buchner  invented  a  method  by  which,  by  the  use  of 
pyrogallic  acid,  the  oxygen  was  absorbed  from  the  atmo- 
sphere in  which  the  culture  was  kept,  and  the  growth 
allowed  to  continue  in  the  nitrogen  and  carbonic  acid 

130 


CULTIVATION  OF  ANAEROBIC  BACTERIA.     131 

which  remained  (Fig.  32).  His  method  was  to  place  the 
tube  which  had  been  inoculated  in  a  much  larger  outer 
test-tube  containing  alkaline  pyrogallic  acid.  The  large 


FIG.  31.  —  Hesse's 
method  of  making 
anaerobic  cultures. 


FIG.  32.— Buchner's 
method  of  making  an- 
aerobic cultures. 


FIG.  33. — Frankel's  meth- 
od of  making  anaerobic  cul- 
tures. 


tube  was  closed  with  a  rubber  cap,  and  the  absorption  of 
the  oxygen  allowed  to  progress. 

Gruber,  instead  of  absorbing  the  oxygen  as  Buchner 
does,  prefers  to  use  an  air-pump  and  exhaust  the  contents 
of  the  tube.  He  uses  a  tube  having  a  slender  neck  and 
a  perforated  rubber  stopper.  After  the  inoculation  is 
made  the  air  is  pumped  out  and  the  slender  neck  sealed 
in  the  blowpipe.  After  this  the  tube  can  be  warmed  and 
the  melted  gelatin  or  agar-agar  rolled  on  its  sides,  as  sug- 
gested by  Ksmarch,  if  desired. 

Better  than  any  of  the  preceding  is  the  method  of 
Frankel,  which  removes  the  air  and  replaces  it  by  hy- 
drogen. Frankel  prepares  an  ordinary  Ksmarch  tube, 
removes  the  cotton  stopper,  and  replaces  it  by  a  carefully 
sterilized  rubber  cork  containing  two  tubes  (Fig.  33).  The 


132  PATHOGENIC  BACTERIA. 

tubes  are  connected  with  a  hydrogen  generator,  and  the 
gas  is  allowed  to  pass  through  until  all  the  oxygen  is 
forced  out  and  replaced  by  the  hydrogen,  after  which  the 
ends  of  the  tubes  are  sealed  in  the  flame  (Fig.  32). 

lyiborius  has  designed  a  special  tube  for  accomplish- 
ing the  same  thing. 

Kitasato  and  Weil  found  the  addition  of  0.3-0.5  per 
cent,  of  sodium  formate  to  be  of  use  in  aiding  the  rapid- 
ity of  the  development  of  anaerobic  cultures.  L,iborius 
found  that  2  per  cent,  of  glucose  added  to  the  culture- 
medium  also  increased  the  rapidity  of  the  process. 

The  methods  now  generally  employed  by  bacteri- 
ologists for  the  anaerobic  cultivations  embrace  all  the 
essentials  of  the  foregoing  methods.  One  of  the  best 
arrangements  for  the  purpose  is  that  devised  by  Dr. 
Ravenel.  His  inoculations  are  deeply  made  in  culture- 
media  as  free  from  air  as  possible.  The  tubes  are 
loosely  plugged,  and  are  placed  in  an  air-tight  cham- 
ber the  bottom  of  which  contains  pyrogallic  acid — py- 
rogallic  acid  i,  solution  of  caustic  potash  i,  water  10. 
The  apparatus  is  connected  by  two  tubes  with  an  ex- 
haust-pump on  one  side,  and  with  a  hydrogen  appara- 
tus on  the  other,  by  which  means  the  atmosphere  is  ex- 
hausted, and  replaced  by  hydrogen  until  only  pure  hydro- 
gen remains,  after  which  the  chamber  is  permanently 
sealed  and  the  germs  allowed  to  grow.  Such  a  chamber 
can  be  constructed  to  hold  a  number  of  tubes  or  Petri 
dishes,  yet  not  be  too  large  to  be  stood  in  an  incubator. 
Whatever  oxygen  may  have  escaped  the  exhaustion  or 
have  entered  by  the  process  of  leakage  is  at  once  absorbed 
by  the  pyrogallic  acid  in  the  lower  chamber  of  the  ap- 
paratus. 

An  apparatus  for  plating  out  strictly  anaerobic  bac- 
teria that  has  met  with  great  favor  is  that  invented  by 
Botkin  (Fig.  34).  It  combines  the  replacement  of  the 
air  by  hydrogen  and  the  absorption  of  the  oxygen  possi- 
bly remaining  by  alkaline  pyrogallic  acid.  In  using  the 
apparatus  the  uncovered  Betri  dishes  are  placed  one 


CULTIVATION  OF  ANAEROBIC  BACTERIA.     133 

above  the  other  in  the  rack  c,  and  covered  with  the  bell- 
glass  A.  Liquid  paraffin  is  poured  in  the  dish  B  until  it 
is  about  half  full.  From  a  Kipp's  apparatus  hydrogen 
gas  enters  the  little  rubber 
tube  #,  subsequently  escap- 
ing by  the  tube  b.  When 
only  pure  hydrogen  escapes 
the  rubber  tubes  a  and  b  are 
withdrawn,  and  the  appa- 
ratus remains  filled  with  hy- 
drogen. Lest  a  little  oxygen 
should  remain,  it  is  best  to 
have  the  dishes  at  the  top 
and  bottom  of  the  neck  filled 
with  alkaline  pyrogallic  acid. 
Tetanus  can  be  cultivated  in 
this  apparatus. 

Roux   has   suggested    the 
simplest  method  of  cultivat-  ^  0  . .  ,  ^    , 

FIG.  34. — Botkm's  apparatus  for  mak- 
ing anaerobic  bacteria.      The        ing  anaerobic  plate- cultures, 
germs  are  distributed  through 

freshly  boiled,  still  liquid,  gelatin  or  agar-agar,  as  in 
making  the  dilutions  for  plate-cultures,  then  drawn  into 
a  long,  slender  sterile  piece  of  glass  tubing  of  small 
calibre.  When  the  tube  is  full  the  ends,  which  should 
have  been  narrowed,  are  closed  in  a  flame,  and  the  cul- 
ture is  hermetically  sealed  in  an  air-tight  chamber.  The 
chief  difficulty  is  in  transplanting  the  growing  colony. 
To  do  this  the  tube  must  be  opened  with  a  file  or  a  dia- 
mond at  the  point  where  the  colony  desired  is  observed. 


CHAPTER  IX. 
EXPERIMENTATION  UPON  ANIMALS. 

BACTERIOLOGY  has  to-day  become  a  science  whose 
principal  objects  are  to  discover  the  cause,  explain  the 
symptoms,  and  prepare  the  cure  of  diseases.  We  can- 
not hope  to  achieve  these  objects  except  by  the  intro- 
duction of  bacteria  into  animals,  where  their  effects  and 
the  effects  of  their  products  can  be  studied. 

No  one  should  more  heartily  condemn  wanton  cruelty 
to  animals  than  the  physician  and  the  naturalist.  In- 
deed, it  is  hard  to  imagine  a  class  of  men  so  much  of 
whose  lives  is  spent  in  relieving  pain,  and  who  know  so 
much  about  pain,  being  guilty  of  the  wholesale  butchery 
and  torture  accredited  to  them  by  a  few  of  the  laity, 
whose  eyes,  but  not  whose  brains,  have  looked  over 
the  pages  of  physiological  text-books. 

Experimentation  upon  animals  has  given  us  almost 
all  our  knowledge  of  physiology,  most  of  our  valuable 
therapeutics,  and  the  only  scientific  methods  of  treating 
tetanus  and  diphtheria. 

Experiments  upon  animals  we  must  make,  and,  as 
animals  differ  in  their  susceptibility  to  diseases,  large 
numbers  and  different  kinds  must  be  employed. 

The  bacteriological  methods  are  not  cruel.  Two  prin- 
cipal modes  of  introducing  bacteria  are  employed  :  the 
subcutaneous  injection  and  the  intravenous  injection. 

Subcutaneous  injections  into  animals  are  made  exactly 
as  hypodermic  injections  are  given  to  man. 

The  intravenous  injections  differ  only  in  that  the  needle 
of  the  syringe  is  introduced  into  a  vein.  This  is  easy  in  a 
large  animal  like  a  horse,  but  is  very  difficult  in  a  small 
animal,  and  wellnigh  impossible  in  anything  smaller  than 

134 


EXPERIMENTATION  UPON  ANIMALS. 


135 


a  rabbit.  Such  injections  when  given  to  rabbits  are  gen- 
erally made  into  the  ear-veins,  as  those  most  conspicuous 
and  accessible  (Fig.  35).  A  peculiar  and  important  fact 
to  remember  is,  that  the  less  conspicuous  posterior  vein 
is  much  better  adapt- 
ed to  the  purpose  than 
the  anterior.  The  in- 
troduction of  the  nee- 
dle should  be  made 
from  the  hairy  surface 
of  the  ear. 

Sometimes  intra- 
abdominal  and  intra- 
pleural  injections  are 
made,  and  in  cases 
where  it  becomes  ne- 
cessary to  determine 
the  presence  or  ab- 
sence of  tuberculosis 

or  glanders  in   tissues       FlG-  35-— Method  of  making  an  intravenous 
.,  r  injection  into  a  rabbit.     Observe  that  the  needle 

it  may  be   necessary  .    .  ,  . 

J  J     enters  the  posterior  vein  from  the  hairy  surface. 

to     introduce     small 

pieces   of  the  suspected  tissue  under  the   skin  or  into 

the  abdominal  cavities. 

Sometimes  the  inoculation  can  be  made  by  the  platinum 
wire,  a  very  small  opening  in  the  skin  being  sufficient. 

Small  animals,  like  rabbits  and  guinea-pigs,  can  be 
held  in  the  hand,  as  a  rule.  Rabbit-holders  of  various 
forms  can  be  obtained  from  dealers.  Dogs,  cats,  sheep, 
and  goats  can  be  tied  and  held  in  troughs.  A  convenient 
form  of  mouse-holder,  invented  by  Kitasato,  is  shown  in 
Figure  36. 

In  all  these  experiments  one  must  remember  that  the 
amount  of  material  introduced  into  the  animal  must  be 
in  proportion  to  its  size,  and  that  injection-experiments 
upon  mice  generally  are  so  crude  and  destructive  as  to 
warrant  the  comparison  drawn  by  Frankel,  that  to  inject 
a  few  minims  of  liquid  into  the  pleural  cavity  of  a  mouse 


136  PATHOGENIC  BACTERIA. 

is  u  much  the  same  as  if  one  would  inject  through  a  fire- 
hose three  or  four  quarts  of  some  liquid  into  the  respira- 
tory organs  of  a  man. ' ' 

The  blood  of  animals,  when  it  is  necessary  to  experi- 
ment with  it,  is  best  secured  from 
a  large  vein,  generally  the  jugu- 
lar. From  small  animals,  such  as 
guinea-pigs,  it  may  be  secured  by 
introducing  a  small  cannula  into 
the  carotid  artery. 

Our  observations  of  animals  by 
no  means  cease  with  their  death. 
Indeed,  he  cannot  be  a  bacteriol- 
ogist who  is  not  already  a  good 
pathologist  and  expert  in  the  recog- 
nition of  diseased  organs. 

When  an  autopsy  is  to  be  made 
upon  a  small  animal,  it  is  best  to 

FIG.  36.-Mouse-holder.  wash  *  for  a  few  moments  in  a 
disinfecting  solution,  to  kill  the 

germs  present  upon  the  hair  and  the  skin,  as  well  as  to 
moisten  the  hair  and  enable  it  to  be  kept  out  of  the 
incision. 

The  animal  should  be  tacked  to  a  board  if  small,  or 
tied,  by  cords  fastened  to  the  legs,  to  the  corners  of  a 
table  if  large,  and  should  be  dissected  with  sterile  knives 
and  scissors.  When  a  culture  is  to  be  made  from  the 
interior  of  an  organ — say  the  spleen — it  should  be  incised 
deeply  with  a  sterile  knife  and  the  culture  made  from 
its  centre. 

Fragments  intended  for  subsequent  microscopical  ex- 
amination should  be  cut  very  small  (cubes  of  i  c.cm.), 
placed  in  absolute  alcohol  for  a  few  hours,  then  trans- 
ferred to  weaker  alcohol,  80-90  per  cent.,  for  preserva- 
tion. The  technique  of  imbedding  and  staining  the  tis- 
sues can  be  found  in  almost  any  reliable  text-book  on 
pathology  or  on  the  special  subject  of  microscopical 
technique. 


CHAPTER    X. 
THE  RECOGNITION  OF  BACTERIA. 

THE  most  difficult  thing  in  bacteriology  is  to  be  able 
to  recognize  the  bacteria  which  come  under  observation. 

A  certain  few  micro-organisms  are  so  characteristic  in 
shape  and  grouping  as  to  be  separated  by  a  microscopic 
examination.  Some,  as  the  tubercle  bacillus,  are  charac- 
teristic in  their  reaction  to  the  anilin  dyes,  and  can  be 
differentiated  at  once  by  this  peculiarity.  Some,  as  the 
Bacillus  mycoides,  are  so  characteristic  in  their  agar-agar 
growth  as  to  eliminate  others.  The  red  color  of  Bacillus 
prodigiosus  and  the  blue  of  Bacillus  janthinus  will  speak 
almost  positively  for  them.  The  potato  culture  of  the 
Bacillus  mesentericus  fuscus  and  its  close  relative  the  vul- 
gatus  is  quite  sufficient  to  enable  us  to  pronounce  upon 
them.  Unfortunately,  however,  there  are  several  hun- 
dreds of  described  species  which  lack  any  one  distinct 
character  that  may  be  used  for  differential  purposes,  and 
require  that  for  their  diagnosis  we  shall  wellnigh  ex- 
haust the  bacteriological  technique  in  an  almost  fruitless 
effort  to  recognize  them. 

A  series  of  useful  tables  has  been  compiled  by  Eisen- 
berg,  and  is  now  almost  indispensable  to  the  worker. 
Unfortunately,  in  tabulating  bacteria  we  constantly  meet 
species  described  so  insufficiently  as  to  make  them  worse 
than  useless  on  account  of  the  confusion  caused. 

The  only  way  to  recognize  a  species  is  to  study  it 
thoroughly  and  compare  it,  step  by  step,  with  the  descrip- 
tions and  tables  of  known  species  compiled  by  Eisenberg 
and  others. 

137 


CHAPTER  XI. 
THE   BACTERIOLOGIC  EXAMINATION  OF  THE  AIR. 

IT  has  been  repeatedly  emphasized — and  indeed  at  the 
present  time  almost  every  one  knows — that  micro-organ- 
isms float  almost  everywhere  in  the  air,  and  that  their 
presence  there  is  a  constant  source  of  danger,  not  only 
of  contamination  in  our  bacteriologic  researches,  but 
also  a  menace  to  our  health. 

Such  micro-organisms  are  neither  ubiquitous  nor  equally 
disseminated,  but  are  much  more  numerous  where  the  air 
is  dusty  than  where  it  is  pure — much  more  so  where  men 
and  animals  are  accustomed  to  live,  than  upon  the  ocean 
or  upon  high  mountain-tops.  The  purity  of  the  atmo- 
sphere bears  a  distinct  relation  to  the  purity  of  the  soil 
over  which  its  currents  blow. 

The  micro-organisms  that  occur  in  the  air  are  for  the 
most  part  harmless  saprophytes  which  have  been  sepa- 
rated from  their  nutrient  birthplace  and  carried  about  by 
the  wind.  They  are  almost  always  taken  up  from  dried 
materials,  experiment  having  shown  that  they  arise  from 
the  surfaces  of  liquids  in  which  they  grow  with  much  dif- 
ficulty. They  are  by  no  means  all  bacteria,  and  a  plate 
of  sterile  gelatin  exposed  for  a  brief  time  to  the  air  will 
generally  grow  moulds  and  yeasts  as  well  as  bacteria. 

The  bacteria  present  are  occasionally  pathogenic,  espe- 
cially in  localities  where  the  discharges  of  diseased  animals 
have  been  allowed  to  collect  and  dry.  For  this  reason  the 
atmosphere  of  the  wards  of  hospitals  and  of  rooms  in 
which  infectious  cases  are  being  treated  is  much  more 
apt  to  contain  them  than  the  air  of  the  street.  However, 
the  dried  expectoration  of  cases  of  tuberculosis,  of  in- 

138 


BACTERIOLOGIC  EXAMINATION  OF  AIR.       139 

fluenza,  and  sometimes  of  pneumonia,  causes  the  specific 
bacteria  of  these  diseases  to  be  far  from  uncommon  in 
street-dust. 

Giinther  points  out  that  the  majority  of  the  bacteria 
which  occur  in  the  air  are  cocci,  sarcina  being  very 
abundant.  Most  of  them  are  chromogenic  and  do  not 
liquefy  gelatin.  It  is  unusual  to  find  a  considerable 
variety  of  bacteria  at  a  time  ;  generally  not  more  than 
two  or  three  species  are  found. 

It  is  an  easy  matter  to  determine  whether  bacteria  are 
present  in  the  air  or  not,  all  that  is  necessary  being  to 
expose  sterile  plates  or  Petri  dishes  of  gelatin  to  the  air 
for  a  while,  close  them,  and  observe  whether  or  not  bac- 
teria grow  upon  them. 

To  make  a  quantitative  estimation  is,  however,  much 


FIG.  37. — Hesse's  apparatus  for  collecting  bacteria  from  the  air. 

more  difficult.     Several  methods  have  been  suggested,  of 
which  the  most  important  may  be  considered. 

The  method  suggested  by  Hesse  is  simple  and  good. 
It  consists  in  making  a  measured  quantity  of  the  air  to 


1 40  PA  THOGENIC  BA  CTERIA . 

be  examined  pass  through  a  horizontal  sterile  tube  about 
70  cm.  long  and  3.5  cm.  wide  (Fig.  37),  the  interior  of 
which  is  coated  with  gelatin  in  the  same  manner  as  an 
Esmarch  tube.  The  tube,  having  been  prepared,  is 
closed  at  both  ends  with  sterile  corks  carrying  smaller 
glass  tubes  closed  with  cotton.  When  ready  for  use  the 
tube  at  one  end  is  attached  to  a  hand-pump,  the  cotton 
is  removed  from  the  other  end,  and  the  air  passed  through 
very  slowly,  the  bacteria  having  time  to  precipitate  upon 
the  gelatin  as  they  pass.  When  the  required  amount  has 
passed  the  tubes  are  again  plugged,  the  apparatus  stood 
away  for  a  time,  and  subsequently,  when  they  have 
grown,  the  colonies  are  counted.  The  number  of  colo- 
nies in  the  tube  will  represent  pretty  accurately  the 
number  of  bacteria  in  the  amount  of  air  which 
passed  through  the  tube. 

In  such  a  cylindrical  culture  it  will  be  noted 
that  if  the  air  is  passed  through  with  the 
proper  slowness,  the  colonies  will  be  much 
more  numerous  near  the  end  of  entrance  than 
that  of  exit.  The  first  to  fall  will  probably 
be  those  of  heaviest  specific  gravity — i.  e.  the 
moulds  and  yeasts. 

A  still  more  exact  method  is  that  of  Petri, 
who  uses  small  filters  of  sand  held  in  place  in  a 
wide  glass  tube  by  small  wire  nets  (Fig.  38). 
The  sand   used  is  made   to  pass   through  a 
sieve  whose  openings  are  of  known  size,   is 
heated   to   incandescence,    then   arranged    in 
the  tube  so  that  two  of  the  little  filters,  held 
in   place   by  their  wire-gauze  coverings,  are 
FIG.  38.—   superimposed.     One  or  both  ends  of  the  tube 
etn  s    san     ^^  closed  with  corks  having-  a  narrow  glass 

filter  for  air-  * 

examination,  tttbc.  The  apparatus  is  heated  and  sterilized 
in  a  hot-air  sterilizer,  and  is  then  ready  for 
use.  The  method  of  employment  is  very  simple.  By 
means  of  a  hand-pump  100  liters  of  air  are  made  to  pass 
through  in  from  ten  to  twenty  minutes.  The  sand  from 


BACTERIOLOGIC  EXAMINATION  OF  AIR.      141 


the  upper  filter  is  then  carefully  mixed  with  sterile 
melted  gelatin  and  poured  into  sterile  Petri  dishes,  where 
the  colonies  develop  and  can  be  counted.  Sternberg  re- 
marks that  the  chief  objection  to  the  method  is  the  pres- 
ence in  the  gelatin  of  the  slightly  opaque  sand,  which 
interferes  with  the  recognition  and  count- 
ing of  the  colonies.  This  objection  has, 
however,  been  removed  by  Sedgwick  and 
Miquel,  who  use  a  soluble  material — granu- 
lated or  pulverized  sugar — instead  of  the 
sand.  The  apparatus  used  for  the  sugar- 
experiments  differs  a  little  from  the  original 
of  Petri,  but  the  principle  is  the  same,  and 
can  be  modified  to  suit  the  experimenter. 
Petri  points  out  in  relation  to  his  method 
that  the  filter  catches  a  relatively  greater 
number  of  bacteria  in  proportion  to  moulds 
than  the  Hesse  apparatus,  which  depends 
upon  sedimentation. 

A  particularly  useful  form  of  apparatus 
is  a  granulated  sugar-filter  suggested  by 
Sedgwick  and  Tucker,  which  has  an  ex- 
pansion above  the  filter,  so  that  as  soon  as 
the  sugar  is  dissolved  in  the  melted  gela- 
tin it  can  be  rolled  out  into  a  lining  like 
that  of  an  Bsmarch  tube.  This  cylindrical 
expansion  is  divided  into  squares  which 
make  the  counting  of  the  colonies  very  easy 
(Fig.  39). 

\      o    oy / 

The  number  of  germs  in  the  atmosphere  FlG-  39-— Sedg- 
will  naturally  be  very  variable.  Roughly,  wiuck>s,  exPanded 

J  fe      J '    tube     for     air-e^- 

the  number  may  be  estimated  at  from  100  animation, 
to  1000  per  cubic  meter. 

In  reality,  the  bacteriologic  examination  of  air  is 
of  very  little  value,  as  so  many  possibilities  of  error 
may  occur.  Thus,  when  the  air  of  a  room  is  quiescent 
there  may  be  very  few  bacteria  in  it ;  let  some  one  walk 
across  the  floor  and  dust  at  once  rises,  and  the  number 


142  PA  THOGENIC  BA  CTERIA. 

of  bacteria  is  considerably  increased :  if  the  person  be  a 
woman  with  skirts,  more  bacteria  will  probably  be  raised 
from  the  floor  than  would  be  disturbed  by  a  man  ;  if  the 
room  be  swept,  the  increase  is  enormous.  From  these 
and  similar  contingencies  it  becomes  very  difficult  to 
know  just  when  and  how  the  air  is  to  be  examined, 
and  the  value  of  the  results  is  correspondingly  lessened. 
The  most  valuable  examinations  are  those  which  aim 
at  the  discovery  of  some  definite  organism  or  organisms 
regardless  of  the  number  per  cubic  meter. 


CHAPTER  XII. 
BACTERIOLOGIC  EXAMINATION  OF  WATER. 

UNLESS  water  has  been  specially  sterilized  or  distilled 
and  received  and  kept  in  sterile  vessels,  it  always  con- 
tains some  bacteria.  The  number  will  bear  a  very  dis- 
tinct relation  to  the  amount  of  organic  matter  in  the 
water,  though  experiment  has  shown  that  certain  patho- 
genic and  non-pathogenic  bacteria  can  remain  vital  in 
perfectly  pure  distilled  water  for  a  considerable  length  of 
time.  Ultimately,  owing  to  the  lack  of  nutriment,  they 
undergo  a  granular  degeneration. 

The  majority  of  the  water-bacteria  are  bacilli,  and  as  a 
rule  they  are  non-pathogenic.  Of  course,  at  times  the 
most  virulent  forms  of  pathogenic  bacteria — those  of 
cholera  and  typhoid  fever — occur  in  polluted  water,  but 
this  is  the  exception,  not  the  rule. 

The  method  of  determining  quantitatively  the  number 
of  bacteria  in  water  is  very  simple,  and  can  generally  be 
prosecuted  without  much  apparatus.  The  principle  de- 


FlG.  40. — Wolf  hiigel's  apparatus  for  counting  colonies  of  bacteria  upon  plates. 


pends  upon  the  equal  distribution  of  a  given  quantity  of 
the  water  to  be  examined  through  a  sterile  liquid  medium, 
and  the  subsequent  solidification  of  this  medium  in  a 

143 


i44 


PATHOGENIC  BACTERIA. 


thin  layer,  so  that  all  the  colonies  which  develop  may 
be  counted. 

The  method,  which  originated  with  Koch,  may  be  per- 
formed with  the  Koch  plates  or  with  Petri  dishes  or 
with  Bsmarch  rolls.  It  is  always  best  to  make  a  num- 
ber of  these  plate-cultures  with  different  amounts  of  the 
water  to  be  examined,  using,  for  example,  o.oi,  o.i,  0.5, 
and  i.o  c.cm.  added  to  a  tube  of  gelatin,  agar-agar,  or 
glycerin  agar-agar. 

The  exact  method  must  depend  somewhat  upon  the 
quality  of  the  water  to  be  examined.  If  the  number  of 
bacteria  per  cubic  centimeter  is  small,  large  quantities 
may  be  used,  but  if  there  are  millions  of  bacteria  in 
every  cubic  centimeter,  it  may  be  necessary  to  dilute  the 
water  to  be  examined  in  the  proportion  of  i  :  10  or  i  :  100 
with  sterile  water,  mixing  well,  and  making  the  plate- 
cultures  from  the  dilutions. 


FIG.  41. — Heyroth's  instrument  for  counting  colonies  of  bacteria  in  Petri  dishes. 

It  is  best  to  count  all  the  colonies  if  possible,  but  when 
there  are  hundreds  or  thousands  scattered  over  the  plate, 


BACTERIOLOGIC  EXAMINATION  OF   WATER.   145 

an  average  estimation  of  a  number  of  squares  ruled  upon 
a  glass  background  (Fig.  40),  as  suggested  by  Wolf  hiigel, 
is  most  convenient.  In  his  apparatus  a  large  plate  of  glass 
is  divided  into  small  square  di- 
visions, the  diagonals  being  spe- 
cially indicated  by  color.  The 
plate  or  Petri  dish  is  stood  upon 
the  glass,  and  the  number  of 
colonies  in  a  number  of  small 
squares  is  easily  counted,  and 
the  total  number  of  colonies  es- 
timated. In  counting  the  colo- 
nies a  lens  is  indispensable. 
Special  apparatuses  have  been 
devised  for  counting  the  colo- 
nies in  Petri  dishes  (Fig.  4l)  ,  FlG*  42-Esmarch's  instrument 
^  °  ~  '  for  counting  colonies  of  bactena 

and  in  Ksmarch  tubes  (Fig.  42).   -m  tUDes. 

The   majority  of  the   water- 
bacteria  are  rapid  liquefiers  of  gelatin,  for  which  reason 
it  seems  better  to  employ  agar-agar  than  gelatin  for 
making  the  cultures. 

In  ordinary  hydrant-water  the  bacteria  number  from 
2-50  per  cubic  centimeter ;  in  good  pump- water,  100-500 ; 
in  filtered  water  from  rivers,  according  to  Gunther,  50-200 
are  present ;  in  unfiltered  river- water,  6000-20,000.  Ac- 
cording to  the  pollution  of  the  water  the  number  may 
reach  as  many  as  50,000,000. 

The  waters  of  wells  and  springs  are  dependent  for  their 
purity  upon  the  character  of  the  earth  or  rock  through 
which  they  filter,  and  the  waters  of  deep  wells  are  much 
more  pure  than  those  of  shallow  wells,  unless  contamina- 
tion takes  place  from  the  surface  of  the  ground. 

Ice  always  contains  bacteria  if  the  water  contained 
them  before  it  was  frozen.  In  Hudson-River  ice  Prud- 
den  found  an  average  of  398  colonies  in  a  cubic  centi- 
meter. 

A  sample   of  water  when   collected  for  examination 
should  be  placed  in  a  clean  sterile  bottle  or  in  a  her- 
10 


146  PATHOGENIC  BACTERIA, 

metically-sealed  pre-sterilized  glass  bulb,  and  must  be 
examined  as  soon  as  possible,  as  the  bacteria  multiply 
rapidly  in  water  which  is  allowed  to  stand  for  a  short 
time.  In  determining  the  species  of  bacteria  found  in 
the  water  reference  must  be  made  to  the  numerous  mono- 
graphs upon  the  subject,  and  to  tables  such  as  those 
compiled  by  Eisenberg. 

The  discovery  of  certain  important  pathogenic  bacteria, 
as  those  of  cholera  and  typhoid,  will  be  considered  under 
the  specific  headings. 

Unfortunately,  the  bacteriologic  examination  of  waters 
does  not  throw  satisfactory  light  upon  their  exact  hygi- 
enic usefulness.  Of  course,  if  cholera  or  typhoid-fever 
bacteria  are  present,  the  water  is  harmful,  but  the  quality 
of  the  water  cannot  be  gauged  by  the  number  of  bacteria 
it  contains. 

Filtration  with  sand,  etc.  diminishes  the  number  of 
bacteria  for  a  time,  but,  as  the  organisms  multiply  in 
the  filter,  the  benefit  is  not  permanent.  The  filters  must 
frequently  be  renewed.  Porcelain  filters  seem  to  be  the 
only  positive  safeguard,  and  even  these,  the  best  of  which 
seems  to  be  the  Pasteur-Chamberland,  allow  the  bacteria 
to  pass  through  if  used  too  long  without  renewal  or  with- 
out firing. 


CHAPTER   XIII. 


BACTERIOLOGIC  EXAMINATION  OF  SOIL. 

ALMOST  all  soil  contains  bacteria  in  its  upper  layers. 
Their  number  and  character,  however,  depend  some- 
what upon  the  surrounding  conditions.  Near  the  hab- 
itations of  men,  where  the  soil  is  cultivated,  the  ex- 
crement of  animals,  largely  made  up  of  bacteria,  is 
spread  upon  it  to  increase  its  fertility,  this  being  a  treat- 
ment which  not  only  adds  new  bacteria  to  those  already 
present,  but  also  enables  those  present  to  grow  very  much 
more  luxuriantly  because  of  the  increased 
amount  of  organic  matter  they  receive. 

The  researches  of  Fliigge,  C.  Frankel, 
and  others  show  that  the  bacteria  of  the 
soil  do  not  penetrate  very  deeply — that 
they  gradually  decrease  in  number  until 
the  depth  of  a  meter  is  reached,  then 
rapidly  diminish  until  at  a  meter  and  a 
quarter  they  rather  abruptly  cease  to  be 
found. 

Many  of  the  soil-bacteria  are  anaerobic, 
and  for  a  careful  consideration  of  them 
the  reader  must  be  referred  to  monographs 
upon  the  subject.  The  estimation  of  their 
number  seems  to  be  devoid  of  any  dis- 
tinct practical  importance.  C.  Frankel 
has,  however,  originated  a  very  accurate 
method  of  determining  it.  By  means 
of  a  special  boring  apparatus  (Fig.  43) 
earth  can  be  secured  from  any  depth  without  digging  and 
without  danger  of  mixing  that  secured  with  that  of  the 
superficial  strata.  With  sterile  liquefied  gelatin  a  definite 

147 


FIG.  43.— Fran- 
kel's  instrument  for 
obtaining  earth  from 
various  depths  for 
bacteriologic  study. 


148  PATHOGENIC  BACTERIA. 

amount  of  this  soil  is  mixed  thoroughly  and  the  mixture 
solidified  upon  the  walls  of  an  Bsmarch  tube.  The  col- 
onies are  counted  with  the  aid  of  a  lens.  Fliigge  found 
in  virgin  earth  about  100,000  colonies  in  a  cubic  centi- 
meter. 

Samples  of  earth,  like  samples  of  water,  should  be 
examined  as  soon  as  possible  after  being  secured,  for, 
as  Giinther  points  out,  the  number  of  bacteria  changes 
because  of  the  unusual  environment,  exposure  to  increased 
amounts  of  oxygen,  etc. 

The  most  important  bacteria  of  the  soil  are  those  of 
tetanus  and  malignant  edema,  in  addition  to  which,  how- 
ever, there  are  a  great  variety  which  are  pathogenic  for 
rabbits,  guinea-pigs,  and  mice. 


PART  II.     SPECIFIC  DISEASES  AND  THEIR 
BACTERIA. 


A.     THE   PHLOGISTIC   DISEASES. 


I.     THE   ACUTE   INFLAMMATORY  DISEASES. 


CHAPTER    I. 
SUPPURATION. 

SUPPURATION  was  at  one  time  supposed  to  be  an 
inevitable  outcome  of  the  majority  of  wounds,  and, 
although  bacteria  were  observed  in  the  discharges,  the 
old  habit  of  thought  and  insufficiency  of  information 
caused  most  surgeons  to  believe  that  they  were  sponta- 
neously developed  there. 

Sir  Joseph  Leister,  whose  name  we  cannot  sufficiently 
honor,  conceived  that  Pasteur's  observations  upon  the 
germs  of  life  floating  in  the  atmosphere,  if  they  explained 
the  contamination  of  his  sterile  infusions,  might  also 
explain  the  changes  in  wounds,  and  upon  this  idea 
based  that  most  successful  system  of  treatment  known 
as  ''antiseptic  surgery." 

The  further  development  of  antiseptic  surgery,  and  the 
extremes  into  which  it  was  carried  by  alienists,  almost 
attain  to  the  ridiculous,  for  not  only  were  the  hands  of 
the  operator,  his  instruments,  sponges,  sutures,  ligatures, 
and  dressings  kept  constantly  saturated  with  irritating 
germicidal  solutions,  but  at  one  time  the  air  over  the 
wound  was  carefully  saturated  with  pulverized  antiseptic 
lotions  during  the  whole  operation  by  means  of  a  steam 
atomizer.  This  rather  monstrous  outcome  of  the  appli- 
cation of  Lister's  system  to  surgery  was  the  very  natural 
result  of  the  erroneous  idea  that  the  germs  which  cause 

149 


150  PATHOGENIC  BACTERIA. 

the  suppurative  changes  in  wounds  entered  the  exposed 
tissues  principally  from  the  atmosphere,  and  that  the 
hands  and  instruments  of  the  operator,  while  certainly 
means  of  infection,  were  secondary  in  importance  to  it. 

The  researches  of  more  recent  date,  however,  have 
shown  not  only  that  the  atmosphere  cannot  be  disin- 
fected, but  also  that  the  air  of  ordinarily  quiet  rooms, 
while  containing  the  spores  of  numerous  saprophytic 
organisms,  very  rarely  contains  many  pathogenic  bac- 
teria. We  now  also  know  that  a  direct  stream  of  air, 
such  as  is  generated  by  an  atomizer,  causes  more  bacteria 
to  be  conveyed  into  a  wound  than  would  ordinarily  fall 
upon  it,  thereby  increasing  instead  of  lessening  the  dan- 
ger of  infection.  It  may  therefore  be  stated,  with  a 
reasonable  amount  of  certainty,  that  the  atmosphere  is 
rarely  an  important  factor  in  the  process  of  suppuration. 

We  have  already  called  attention  to  the  fact  that 
various  micro-organisms  are  so  intimate  in  their  relation 
to  the  skin  that  it  is  almost  impossible  to  get  rid  of  them, 
and  have  cited  in  this  relation  the  experiments  of  Welch, 
Robb,  and  Ghriskey,  whose  method  of  disinfecting  the 
hands  has  been  recommended  as  the  best.  The  investi- 
gations of  these  observers  have  shown  that,  no  matter 
how  rigid  the  disinfection  of  the  patient's  skin,  the 
cleansing  of  the  operator's  hands,  the  sterilization  of 
the  instruments,  and  the  precautions  exercised,  a  certain 
number  of  wounds  in  which  sutures  are  employed  will 
always  suppurate.  The  cause  of  the  suppuration  is  a 
matter  of  vast  importance  in  surgery  and  in  surgical  bac- 
teriology, yet  it  is  one  which  it  is  impossible  to  remove. 
We  carry  it  constantly  with  us  upon  our  skins. 

Welch  has  described,  under  the  name  Staphylococcus 
epidermidis  albus,  a  micrococcus  which  seems  to  be  habit- 
ually present  upon  the  skin,  not  only  upon  the  surface, 
but  also  deep  down  in  the  Malpighian  layer.  He  is  of 
the  opinion  that  it  is  the  same  organism  which  is  familiar 
to  us  under  the  name  of  Staphylococcus  pyogenes  albus, 
but  in  an  attenuated  condition.  If  his  opinion  be  correct, 


SUPPURATION.  151 

and  we  have  seated  deeply  in  our  derm  a  coccus  which 
can  at  times  cause  abscess-formation,  the  conclusions  of 
Robb  and  Ghriskey,  that  sutures  of  catgut  when  tightly 
drawn  may  be  a  cause  of  skin-abscesses  by  predisposing 
to  the  development  of  this  organism,  are  certainly  justi- 
fiable. 

Not  only  does  the  coccus  occur  in  the  attenuated  form 
described,  but  we  have  very  commonly  present  upon  the 
skin,  generally  as  a  harmless  saprophyte,  the  important 
Staphylococcus  pyogenes  albus,  which  is  a  common  cause 
of  suppuration. 

Although,  as  stated,  the  Staphylococcus  pyogenes  albus 
is  a  common  cause  of  suppuration,  it  rarely  occurs  alone, 
the  studies  of  Passet  showing  that  in  but  4  out  of  33  cases 
which  he  investigated  was  this  coccus  found  by  itself. 
When  pure  cultures  of  the  coccus  are  injected  subcu- 
taneously  into  rabbits  and  guinea-pigs,  abscesses  some- 
times result ;  sometimes  there  is  no  result.  Injected 
into  the  circulation  of  these  animals,  the  staphylococci 
sometimes  cause  septicemia,  and  after  death  can  be  found 


FIG.  44. — Staphylococcus  pyogenes  aureus,  from  an  agar-agar  culture;   x  1000 

(Gunther). 

in  the  capillaries,  especially  of  the  kidneys.  From  these 
illustrations  it  will  be  seen  that  the  organism  is  feebly 
pathogenic. 


152  PATHOGENIC  BACTERIA. 

In  the  characteristics  of  its  growth  the  Staphylococcus 
albus  is  almost  identical  with  the  species  next  to  be  de- 
scribed, but  differs  from  it  in  that  there  is  no  golden 
color  produced.  Upon  the  culture-media  it  grows  white. 

Generally  present  upon  the  skin,  though  in  smaller 
numbers,  is  the  dangerous  and  highly  virulent  Staphylo- 
coccus pyogenes  aureus  (Fig.  44),  or  "  golden  Staphylococ- 
cus" of  Rosenbach.  As  the  morphology  of  this  organ- 
ism, and  indeed  the  generality  of  its  characters,  are 
identical  with  those  of  the  preceding  species,  it  seems 
convenient  to  describe  them  together,  pointing  out  such 
differences  as  occur  step  by  step.  In  doing  this,  how- 
ever, it  must  not  be  forgotten  that,  although  the  Staphy- 
lococcus albus  has  been  described  first,  the  Staphylococcus 
aureus  is  the  more  common  organism  of  the  suppurative 
diseases. 

Although  they  had  been  seen  earlier  by  several  ob- 
servers, the  staphylococci  were  not  isolated  and  care- 
fully described  until  1884,  when  Rosenbach  worked  upon 
them.  The  results  of  his  study,  followed  by  Passet  and 
a  host  of  others,  have  now  given  us  pretty  accurate 
information  about  them. 

The  cocci  are  distributed  rather  sparingly  in  nature, 
seeming  not  to  find  a  purely  saprophytic  existence  a 
suitable  one.  They  occur,  however,  wherever  man  and 
animals  have  been,  and  can  be  found  in  the  dust  of 
houses,  hospitals,  and  especially  surgical  wards  where 
proper  precautions  are  not  exercised.  They  are  common 
upon  the  skin,  they  live  in  the  nose,  mouth,  eyes,  and 
ears  of  man,  they  are  nearly  always  beneath  the  finger- 
nails, and  they  sometimes  occur  in  the  feces,  especially 
in  children. 

The  cocci  are  rather  small,  measuring  about  o.  7  p  in 
diameter.  When  examined  in  a  delicately-stained  con- 
dition the  organisms  may  be  seen  to  consist  of  hemi- 
spheres separated  from  each  other  by  a  narrow  interval. 
The  contiguous  surfaces  are  flat,  thus  differing  from 
the  gonococcus,  whose  contiguous  surfaces  are  concave. 


SUPPURA  TION.  153 

The  grouping  is  not  very  characteristic.  In  both  liquid 
and  solid  culture-media  the  organisms  either  occur  in 
solid  masses  or  are  evenly  distributed.  It  is  only  in  the 
organs  or  tissues  of  a  diseased  animal  that  it  is  possible  to 
say  that  a  true  staphylococcus  grouping  is  present. 

The  organism  stains  brilliantly  with  aqueous  solu- 
tions of  the  anilin  dyes.  In  tissues  it  can  be  beautifully 
stained  by  Gram's  method. 

The  staphylococci  grow  well  either  in  the  presence  or 
absence  of  oxygen  at  a  temperature  above  18°  C.,  the 
most  rapid  development  being  at  about  37°  C.  Upon  the 
surface  of  gelatin  plates  small  whitish  points  can  be 
observed  in  forty-eight  hours  (Fig.  45).  These  rapidly 


FIG.  45. — Staphylococcus  pyogenes  aureus :  colony  two  days  old,  seen  upon  an 
agar-agar  plate ;    x  40  ( Heim) . 

extend  to  the  surface  and  cause  extensive  liquefaction. 
Hand  in  hand  with  the  liquefaction  is  the  formation  of 
an  orange  color,  which  is  best  observed  at  the  centre  of 
the  colony.  Under  the  microscope  the  colonies  appear 
as  round  disks  with  circumscribed,  smooth  edges.  They 
are  distinctly  granular  and  dark-brown.  When  the  col- 
onies are  grown  upon  agar-agar  plates  the  formation  of 
the  pigment  is  much  more  distinct. 

In  gelatin  punctures  the  growth  occurs  along  the  whole 
length  of  the  needle-track,  and  causes  an  extensive  lique- 


154 


PATHOGENIC  BACTERIA. 


faction  in  the  form  of  a  long,   narrow,   blunt-pointed, 
inverted  cone  (Fig.  46)  full  of  clouded  liquid,  at  the  apex 


FIG.  46. — Staphylococcus  pyogenes  aureus :   puncture-culture  three  days  old 
in  gelatin  (Frankel  and  Pfeiffer). 

of  which  a  collection  of  golden  or  orange-yellow  precipi- 
tate is  always  present.  It  is  this  precipitate  in  particu- 
lar that  gives  the  organism  its  name,  "golden  staphylo- 
coccus." 

The  most  characteristic  growth  is  upon  agar-agar. 
Along  the  whole  line  of  inoculation  an  orange-yellow, 
moist,  shining  growth  occurs.  When  the  growth  takes 
place  rapidly,  as  in  the  incubator,  it  exceeds  the  rapidity 
of  color-production,  so  that  the  centre  of  the  growth  is 
distinctly  golden  ;  the  edges  may  be  white. 

Upon  potato  the  growth  is  luxuriant,  producing  an 
orange-yellow  coating  over  a  large  part  of  the  surface. 
The  potato-cultures  give  off  a  sour  odor. 

When  grown  in  bouillon  the  organism  causes  a  diffuse 
cloudiness. 


SUPPURA  TION.  1 55 

In  milk  coagulation  takes  place,  and  is  followed  by 
gradual  digestion  of  the  casein. 

The  Staphylococcus  albus  is  exactly  the  same  as  the 
aureus,  with  the  exception  that  in  all  media  it  is  con- 
stantly colorless. 

Experiments  have  shown  that  the  Staphylococcus 
aureus,  like  its  congener,  the  albus,  exists  in  an  atten- 
uated form,  and  there  is  every  reason  to  believe  that  in 
the  majority  of  instances  it  inhabits  the  surface  of  the 
body  in  this  form. 

When  virulent  the  golden  Staphylococcus  is  a  danger- 
ous and  often  deadly  organism.  Its  pathogeny  among 
animals  is  decided.  When  introduced  subcutaneously, 
abscesses  almost  invariably  follow,  except  in  a  certain 
few  comparatively  immune  species,  and  not  infrequently 
lead  to  a  fatal  termination.  In  such  cases  the  organisms 
may  be  cultivated  from  the  blood  of  the  large  vessels, 
though  by  far  the  greater  number  collect  in,  and  fre- 
quently obstruct,  the  capillaries.  In  the  lungs  and 
spleen,  and  still  more  frequently  in  the  kidneys,  infarcts 
are  formed  by  the  bacterial  emboli.  The  Malpighian 
tufts  of  the  kidneys  sometimes  are  full  of  cocci,  and 
become  the  centres  of  small  abscesses. 

The  coccus  is  almost  equally  pathogenic  for  man, 
though  the  fatal  outcome  is  much  more  rare.  It  enters 
the  system  through  scratches,  punctures,  or  abrasions, 
and  when  virulent  generally  causes  an  abscess,  as  various 
experimenters  who  inoculated  themselves  have  discov- 
ered to  their  cost.  Garre  applied  the  organism  in  pure 
culture  to  the  uninjured  skin  of  his  arm,  and  in  four 
days  developed  a  large  carbuncle  with  a  surrounding 
zone  of  furuncles.  Bockhart  suspended  a  small  portion 
of  an  agar-agar  culture  in  salt-solution,  and  scratched  it 
gently  into  the  deeper  layers  of  the  skin  with  his  finger- 
nail ;  a  furuncle  developed.  Bumm  injected  the  coccus 
suspended  in  salt-solution  beneath  his  skin  and  that  of 
several  other  persons,  and  produced  an  abscess  in  every 
case. 


1 56  PA  THOGENIC  BA  CTERIA . 

The  Staphylococcus  aureus  is  not  only  found  in  the 
great  majority  of  furuncles,  carbuncles,  abscesses,  and 
other  inflammatory  diseases  of  the  surface  of  the  body, 
but  also  plays  an  important  role  in  a  number  of  deeply- 
seated  diseases  of  the  internal  organs.  Becker  and  others 
obtained  it  from  the  pus  of  osteomyelitis,  demonstrating 
that  if,  after  fracturing  or  crushing  a  bone,  the  staphylo- 
coccus  was  injected  into  the  circulation,  osteomyelitis 
would  result.  Numerous  bacteriologists  have  demon- 
strated its  presence  in  ulcerative  endocarditis.  Rodet 
has  been  able  to  produce  osteomyelitis  without  previ- 
ous injury  to  the  bones ;  Rosenbach  was  able  to  produce 
ulcerative  endocarditis  by  injecting  some  of  the  staphy- 
lococci  into  the  circulation  in  animals  whose  cardiac 
valves  had  been  injured  by  a  sound  passed  into  the 
carotid  artery  ;  and  Ribbert  has  shown  that  the  injection 
of  cultures  of  the  organism  may  cause  the  valvular  lesion 
without  the  preceding  injury. 

The  Staphylococcus  aureus  is  an  easy  organism  to  ob- 


FIG.  47. — Streptococcus  pyogenes,  from  the  pus  taken  from  an  abscess ;    x  1000 
(Frankel  and  Pfeiffer). 

tain,  and  can  be  secured  by  plating  out  a  drop  of  pus  in 
gelatin  or  in  agar-agar.     Such  a  preparation,  however, 


SUPPURATION. 


157 


generally  does  not  contain  the  Staphylococcus  aureus 
alone,  but.  shows  colonies  of  the  Staphylococcus  albus  as 
well.  In  addition  to  these  two  principal  forms,  one 
sometimes  discovers  an  organism  identical  with  the  pre- 
ceding, except  that  its  growth  on  agar-agar  and  potato 
is  of  a  brilliant  lemon-yellow  color,  and  its  pathogeny  for 
animals  much  less.  This  is  the  Staphylococcus  citreus  of 
Passet.  It  is  not  quite  so  common,  and  not  so  patho- 
genic as  the  others,  and  consequently  much  less  important. 

Another  organism  whose  colonies  are,  frequently  ob- 
tained from  the  pus  containing  the  staphylococci  is  the 
Streptococcus  pyogenes  of  Rosenbach  (Fig.  47).  It  was 
found  by  him  in  18  of  33  cases  studied, 
fifteen  times  alone  and  five  times 
with  the  Staphylococcus  aureus.  It 
is  a  spherical  organism  of  variable 
size  (0.4-1  fi.  in  diameter),  constantly 
associated  in  pairs  and  chains  of  from 
four  to  twenty  individuals. 

The  organism  stains  well  with  or- 
dinary aqueous  solutions  of  the  anilin 
dyes,  and  also  by  Gram's  method. 
Like  the  coccus  already  described,  it 
is  not  motile  and  does  not  seem  to 
form  spores,  though  sometimes  a  large 
individual  —  much  larger  than  the 
others  in  its  chain — may  be  observed, 
and  may  suggest  the  thought  of  arthro- 
sporulation. 

Upon  gelatin  plates  very  small  col- 
onies of  translucent  appearance  are 
observed.  When  superficial,  they 
spread  out  to  form  flat  disks  about 
o.  5  mm.  in  diameter.  The  microscope 
shows  them  to  be  irregular  and  gran- 
ular, to  have  a  slightly  yellowish  color, 
and  to  have  numerous  irregularities  around  the  edges,  due 
to  projecting  chains  of  the  cocci.  No  liquefaction  occurs. 


FIG.  48. — Streptococ- 
cus pyogenes :  culture 
upon  agar-agar  two  days 
old  (Frankel  and  Pfeif- 
fer). 


158  PATHOGENIC  BACTERIA. 

In  gelatin  puncture-cultures  no  liquefaction  is  observed. 
The  minute  spherical  colonies  grow  along  the  whole 
needle-track  and  form  a  slightly  opaque  granular  line. 

Upon  agar-agar  an  exceedingly  delicate  transparent 
growth  develops  slowly  along  the  line  of  inoculation. 
It  consists  of  almost  transparent,  colorless  small  colo- 
nies which  do  not  become  confluent. 

The  growth  upon  blood-serum  much  resembles  that 
upon  agar-agar.  The  streptococcus  does  not  grow  upon 
potato. 

The  organism  seems  to  grow  well  in  milk  which  is 
coagulated  and  digested. 

The  Streptococcus  is  not  very  sensitive  to  acids,  and 
can  be  grown  quite  well  in  media  with  a  slightly  acid 
reaction. 

Sternberg  found  that  the  streptococci  succumb  to  a 
temperature  of  52-54°  C.  continued  for  ten  minutes. 

The  streptococcus  pyogenes  is  not  very  pathogenic  for 
animals.  Subcutaneous  injections  into  mice  and  rabbits 
are,  as  a  rule,  without  either  general  or  local  manifesta- 
tions of  importance.  If,  however,  an  ear  of  a  rabbit  is 
inoculated  with  a  small  amount  of  a  pure  culture  care- 
fully scratched  in,  a  small  patch  resembling  erysipelas 
usually  results.  The  disturbance  passes  away  in  a  few 
days  and  the  animal  recovers. 

Like  the  staphylococci,  the  Streptococcus  pyogenes  is 
frequently  associated  with  internal  diseases,  and  has  been 
found  in  ulcerative  endocarditis  and  in  the  uterus  in 
bases  of  infective  puerperal  endometritis.  Its  relation 
to  diphtheria  is  of  interest,  for,  while,  in  all  probability, 
the  great  majority  of  cases  of  pseudo-membranous  angina 
are  caused  by  the  Klebs-Loffler  bacillus,  yet  an  undoubted 
number  of  cases  are  met  with  in  which,  as  in  Prudden's 
24  cases,  no  diphtheria  bacilli  can  be  found,  but  which 
seem  to  be  caused  by  a  streptococcus  exactly  resembling 
that  under  consideration. 

There  is  no  clinical  difference  in  the  picture  of  the 
throat-lesion  produced  by  the  two  organisms,  and  the 


SUPPURA  TION.  1 59 

only  positive  method  of  diagnosticating  the  one  from 
the  other  is  by  means  of  a  careful  bacteriologic  examina- 
tion. Such  an  examination  should  always  be  made,  as  it 
has  much  weight  in  connection  with  the  treatment.  Of 
course,  in  streptococcus  angina  no  benefit  could  be  ex- 
pected from  the  diphtheria  antitoxic  serum. 

The  streptococcus  of  Rosenbach  is  thought  by  many 
to  be  identical  with  a  streptococcus  described  by  Fehleisen 
as  the  Streptococcus  erysipelatis  (Fig.  49).  The  two  or- 


FIG.  49. — Streptococcus  erysipelatis,  seen  in  a  section  through  human  skin ; 
x  500  (Frankel  and  Pfeiffer) . 

ganisms  have  much  in  common,  but  much  difference  of 
opinion  exists  upon  the  subject  of  their  identity.  It  may 
seem  unwise  to  omit  the  Streptococcus  erysipelatis  as  a 
major  topic  for  discussion,  but  the  similarity  of  the  or- 
ganism to  that  just  described  has  caused  us  to  consider 
them  in  the  same  connection. 

The  streptococci  of  erysipelas  can  be  obtained  in  almost 
pure  culture  from  the  serum  which  oozes  from  a  puncture 
made  in  the  margin  of  an  erysipelatous  patch.  They  are 
small  cocci,  forming  long  chains — generally  from  six  to 


160  PATHOGENIC  BACTERIA. 

ten  individuals,  but  sometimes  reaching  a  hundred  in 
number.  Occasionally  the  chains  can  be  found  collected 
in  tangled  masses.  They  can  be  cultivated  at  the  room- 
temperature,  but  grow  much  better  at  30-37°  C.  They 
are  not  particularly  sensitive  to  the  absence  of  oxygen, 
but  develop  a  little  more  rapidly  in  its  presence. 

The  erysipelas  cocci,  like  the  Streptococcus  pyogenes, 
are  not  motile,  form  no  spores,  and  are  destroyed  by  a 
low  degree  of  heat.  They  stain  well  with  aqueous  solu- 
tions of  anilin  dyes  and  also  by  Gram's  method. 

The  colonies  upon  gelatin  and  the  development  in 
gelatin  tubes,  upon  agar-agar,  and  upon  blood-serum 
are  identical  with  the  descriptions  of  the  Streptococcus 
pyogenes.  No  growth  occurs  on  potato. 

The  growth  in  bouillon  is  generally  luxuriant,  and  in 
a  short  time  causes  the  medium  to  be  filled  with  chains 
of  the  cocci.  As  the  growth  progresses  these  chains 
gather  in  clusters  and  fall  to  the  bottom  as  a  whitish 
granular  precipitate,  above  which  the  liquid  remains 
clear. 

When  injected  into  animals  Fehleisen's  coccus  behaves 
exactly  like  the  Streptococcus  pyogenes. 

Observation  has  shown  that  dire  results  may  follow  the 
entrance  of  this  organism  into  exposed  wounds,  and  that 
it  causes  not  only  local  suppuration,  but  sometimes  a 
general  infection. 

The  empiric  experience  that  the  occasional  accidental 
infection  of  malignant  tumors  with  erysipelas  cocci  was 
followed  by  sloughing  and  subsequent  disappearance  of 
the  tumor,  suggested  inoculation  with  the  Streptococcus 
erysipelatis  as  a  therapeutic  measure.  The  dangerous 
character  of  the  remedy,  however,  caused  many  to  re- 
frain from  its  use,  for  when  one  inoculated  the  living 
erysipelas  germs  into  the  tissues  he  never  could  estimate 
the  exact  amount  of  disturbance  that  would  follow.  The 
difficulty  seems  to  have  been  overcome  by  Coley,  who 
recommends  the  toxin  instead  of  the  living  coccus  for 
injection.  A  virulent  culture  is  obtained,  inoculated 


SUPPURA  TION.  1 6 1 

into  small  flasks  of  slightly  acid  bouillon,  allowed  to 
grow  for  three  weeks,  then  reinoculated  with  Bacillus 
prodigiosus,  allowed  to  grow  for  ten  or  twelve  days  at 
the  room-temperature,  well  shaken  up,  poured  into  bottles 
of  about  f  sss  capacity,  and  rendered  perfectly  sterile  by  an 
exposure  to  from  50-60°  C.  for  an  hour.  It  is  claimed 
that  the  combined  toxins  of  erysipelas  and  prodigiosus 
are  much  stronger  than  the  simple  erysipelas  toxin.  The 
best  effects  are  found  in  cases  of  sarcoma,  where  the 
toxin  causes  a  rapid  necrosis  of  the  tumor  tissue,  which 
can  be  scraped  out  with  an  appropriate  instrument. 
Numerous  cases  are  on  record  in  which  this  treatment 
has  been  most  efficacious;  but,  although  Coley  recom- 
mends it  and  Czerny  still  upholds  it,  the  majority  of  sur- 
geons have  failed  to  secure  the  desired  results. 

Recently  (1895)  considerable  attention  has  been  be- 
stowed upon  the  development  of  anti-streptococcus  se- 
rum, which  is  said  to  act  specifically  upon  cases  of  strep- 
tococcus-infection, both  general  and  local.  Numerous 


FIG.  50.  —  Bacillus  pyocyaneus,  from  an  agar-agar  culture;    x  1000   (Itzerott 

and  Niemann. 


and  Niemann) 

cases  are  upon   record  in   which   the   serum   exerted  a 
beneficial  action,  though  a  case  reported  by  Weatherly 
11 


162  PATHOGENIC  BACTERIA. 

in  which  Marmorek's  serum  was  used  terminated  fatally 
after  rather  distinct  improvement. 

It  would  seem  as  if  an  antiphlogistic  serum  would 
occupy  an  important  place  in  the  future  of  medicine. 

In  some  cases  the  pus  evacuated  from  wounds  exhibits 
a  peculiar  bluish  or  greenish  color,  from  the  presence  of 
the  Bacillus  pyocyaneus  (Figs.  50,  51).  This  is  a  short, 
delicate  bacillus  of  small  size,  frequently  united  in  chains 
of  four  or  six.  It  has  round  ends,  is  actively  motile,  does 
not  form  spores,  and  can  exist  with  or  without  oxygen. 

The  superficial  colonies  upon  gelatin  plates  form  small, 
irregular,  ill-defined  collections,  which  produce  a  fluores- 
cence of  the  neighboring 
gelatin.  The  gelatin  soft- 
ens gradually,  and  about 
five  days  elapse  before 
liquefaction  is  complete. 

The  microscope  shows 
the  colonies  to  be  round, 
coarsely-granulated  masses 


FIG.  51. — Bacillus  pyocyaneus:  colonies  upon  gelatin  (Abbott). 

with  notched  or  filamentous  borders.  They  have  a  yel- 
low-green color.  Upon  the  surface  they  form  a  delicate 
clump  with  a  smooth  surface,  finely  granular,  distinctly 
green  in  the  middle  and  pale  at  the  edges.  The  colonies 
sink  into  the  gelatin  as  the  liquefaction  progresses. 

In  gelatin  puncture-cultures  most  of  the  development 
occurs  at  the  upper  part  of  the  tube,  where  a  deep  saucer 
of  liquefaction  forms.  The  growth  slowly  descends  into 
the  medium,  and  is  the  point  of  origin  of  a  beautiful 
fluorescence.  The  bacterial  growth  sinks  to  the  bottom 
as  it  ages.  At  times  a  delicate  mycoderma  forms  on  the 
surface. 

Upon  agar-agar  the  growth  is  at  first  bright  green, 


SUPPURA  TION.  163 

developing  all  along  the  line  of  inoculation.  The  green 
pigment  (fluorescin)  is  soluble,  and  soon  saturates  the  cul- 
ture-medium and  makes  it  very  characteristic.  As  the 
culture  ages,  or  if  the  medium  upon  which  it  grows 
contains  much  peptone,  a  second  pigment  (pyocyanin)  is 
developed,  and  the  bright  green  fades  to  a  deep  blue- 
green,  dark-blue,  or  in  some  few  cases  to  a  deep  reddish- 
brown. 

Upon  potato  a  luxuriant  greenish,  smeary  layer  is 
produced. 

This  bacillus  is  highly  pathogenic  for  laboratory  ani- 
mals. About  i  c.cm.  of  a  fresh  bouillon  culture,  if  in- 
jected into  the  subcutaneous  tissue  of  a  guinea-pig  or  a 
rabbit,  causes  a  rapid  edema,  a  suppurative  inflammation, 
and  death  in  a  short  time.  The  bacilli  can  be  found  in 
the  blood  and  in  most  of  the  tissues. 

Intraperitoneal  injections  cause  suppurative  peritonitis. 

It  is  interesting  to  observe,  in  passing,  that  this  path- 
ogeny  can  be  set  aside  by  the  immunity  which  develops 


FIG.  52. — Bacillus  pyogenes  foetidus,  from  agar-agar;     x    1000  (Itzerott  and 

Niemann). 

after  a  few  inoculations  with  sterilized  cultures.  These 
are  easily  prepared,  as  the  thermal  death-point  deter- 
mined by  Sternberg  is  56°  C. 


164  PATHOGENIC  BACTERIA. 

The  bacillus  appears  to  be  rather  common  as  a  sapro- 
phyte, and,  as  it  has  been  found  in  the  perspiration, 
probably  is  not  uncommon  upon  the  skin. 

The  unpleasant  odors  that  sometimes  accompany  sup- 
puration are  probably,  in  most  cases,  due  to  the  Bacil- 
lu s  pyogenes  fcetidus  of  Passet  (Fig.  52).  This  organism 
measures  about  1.5  p.  in  length  and  0.5  //  in  breadth.  It 
is  motile  ;  it  probably  does  not  form  spores.  The  colonies 
are  small  white  disks  without  distinctive  features.  In 
gelatin  punctures  a  thin  grayish-white  growth  occurs 
upon  the  surface,  and  surmounts  a  collection  of  spherical 
confluent  colonies  in  the  puncture.  It  does  not  cause 
any  liquefaction. 

Upon  potato  an  abundant  brownish  growth  takes  place. 

The  cultures  all  give  off  an  unpleasant  putrefactive 
odor.  The  organism  is  only  pathogenic  for  mice  and 
guinea-pigs. 

Occasionally  other  organisms  of  minor  importance  are 
found  in  pus.  Most  of  these,  like  the  Bacillus  pyocyaneus 
and  Bacillus  pyogenes  fcetidus,  are  probably  harmless 
saprophytes  accidentally  present,  so  that  it  will  hardly 
be  proper  to  devote  space  to  their  consideration. 

Before  leaving  the  subject  of  suppuration,  however, 
attention  must  be  called  to  several  rather  common  bac- 
teria which  may  at  times  be  the  cause  of  troublesome 
suppuration.  Among  these  are  the  pneumococcus  of 
Frankel  and  Weichselbaum,  the  Bacillus  coli  communis, 
and  the  typhoid  bacillus. 

The  pneumococcus  has  not  infrequently  been  discov- 
ered most  unexpectedly  in  abscesses  of  the  brain  and 
other  deep-seated  organs,  and  seems  to  have  powerful 
chemotactic  powers.  For  a  careful  consideration  of  it 
the  reader  must  be  referred  to  the  chapter  upon  Pneumo- 
nia, where  it  is  considered  in  full. 

The  Bacillus  coli  communis,  which  is  always  present  in 
the  intestine,  seems  at  times  to  enter  the  blood-  or  lymph- 
channels  and  stimulate  suppuration,  and  numerous  cases 
are  on  record  showing  this.  The  points  most  frequently 


SUPPURATION.  165 

attacked  seem  to  be  the  bile-ducts  and  the  vermiform 
appendix,  though  the  significance  of  the  organism  in 
appendicitis  has  no  doubt  been  overrated.  It  has  also 
been  found  in  the  kidney  in  scarlatinal  nephritis,  and 
is  thought  to  be  the  exciting  cause  of  some  cases.  For 
a  more  particular  study  of  this  organism  the  reader  is 
referred  to  the  chapter  on  Typhoid  Fever. 

The  typhoid  bacillus  is  probably  less  frequently  a  cause 
of  suppuration  than  either  of  the  others,  yet  it  seems  to 
be  the  occasional  cause  of  the  purulent  sequelae  of  typhoid 
fever.  A  case  has  recently  been  reported  by  Flexner  in 
which  metastatic  abscesses  were  found  to  be  caused  by  it. 

The  Micrococcus  tetragenus  has  also  been  found  in  the 
pus  of  acute  abscesses  :  it  is  quite  common  in  the  cavities 
of  pulmonary  tuberculosis,  and  may  aid  in  the  destructive 
processes  involved  in  the  general  phthisical  infection. 

Gonorrhea.  —  All  authorities  now  accept  the  "gono- 
coccus"  to  be  the  cause  of  gonorrhea.  It  was  first  ob- 
served in  the  urethral  and  conjunctival  secretions  of  gon- 


•*  ? 


** 


FIG.  53.—  Gonococcus  in  urethral  pus;    x  1000  (Frankel  and  Pfeiffer). 

orrhea  and  purulent  ophthalmia  by  Neisser  in  1879.  The 
organisms  are  of  hemispherical  shape,  arranged  in  pairs, 
so  that  the  inner  surfaces  are  separated  from  each  other 


1 66  PA  THOGENIC  BA  CTERIA . 

by  a  narrow  interval.  Sometimes,  instead  of  pairs  of 
cocci,  fours  are  seen,  the  group  no  doubt  resulting  from 
the  division  of  a  pair. 

The  described  hemispherical  shape  is  not  exactly  cor- 
rect, for  a  good  lens  generally  shows  the  approximated 
surfaces  to  be  somewhat  concave  rather  than  flat.  The 
Germans  see  in  the  organism  a  resemblance  to  their  pop- 
ular biscuit  called  a  "semmel." 

The  gonococcus  is  small,  is  not  motile,  like  other  cocci, 
is  not  provided  with  flagella,  and  does  not  have  spores. 
It  stains  readily  with  all  the  aqueous  anilin  dyes — best 
with  rather  weak  solutions — but  not  by  Gram's  method. 
It  can  be  found  in  the  urethral  discharges  of  gonorrhea 
from  the  beginning  until  the  end  of  the  disease,  though 
in  the  later  days  its  numbers  may  be  outweighed  by  other 
organisms.  The  organisms  are  generally  found  within 
the  pus-cells  (Fig.  53)  or  attached  to  the  surface  of  epi- 
thelial cells,  and  should  always  be  sought  for  as  diagnostic 
of  gonorrhea,  especially  as  urethritis  sometimes  is  caused 
by  other  organisms,  as  the  Bacillus  coli  communis l  and 
the  Staphylococcus  pyogenes. 

The  cultivation  of  the  gonococcus  is  not  an  easy  task, 
but  one  which  requires  considerable  bacteriologic  skill. 
Wertheim  accomplished  it  by  diluting  a  drop  of  the  pus 
in  a  little  liquid  human  blood-serum,  then  mixing  this 
with  an  equal  part  of  melted  2  per  cent,  agar-agar  at  40° 
C. ,  and  pouring  into  Petri  dishes.  As  soon  as  the  media 
became  firm  the  dishes  were  stood  in  the  incubator  at 
37°  C.,  and  in  twenty-four  hours  the  colonies  could  be 
observed.  Those  upon  the  surface  showed  a  dark  centre, 
around  which  a  delicate  granular  zone  could  be  made 
out. 

When  one  of  these  colonies  is  transferred  to  a  tube  of 
human  blood-serum  or  the  above  mixture  obliquely  co- 
agulated, isolated  little  gray  colonies  occur ;  later  these 
become  confluent  and  produce  a  delicate  smeary  layer 

1  Van  der  Pluyn  und  Loag :  Centralbl.  f.  Bakt.  u.  Parasitenk.,  Bd.  xvii.,  Nos. 
7,  8,  Feb.  28,  1895,  p.  233. 


SUPPURATION.  167 

upon  the  medium.  The  main  growth  is  surrounded  by  a 
thin  veil-like  extension  which  gradually  fades  away  into 
the  medium.  A  slight  growth  occurs  upon  the  water  of 
condensation. 

The  gonococci  may  also  be  cultivated  upon  acid  gela- 
tin, as  pointed  out  by  Turro,  upon  gelatin  containing 
acid  urine,  and  also  in  acid  urine  itself,  where  the  gono- 
cocci grow  near  the  surface,  while  the  pus-cocci  which 
may  be  mixed  with  them  sink  deeper  into  the  medium. 

It  is  ordinarily  presumed  that  gonorrhea  cannot  be 
communicated  to  animals,  but  Turro  asserts  that  the 
gonococci  when  grown  upon  acid  gelatin  readily  com- 
municate urethritis  to  dogs,  and  that  no  Iczsio  continui 
is  necessary,  the  simple  introduction  of  the  organisms 
into  the  meatus  sufficing  to  produce  the  disease. 

That  the  gonococcus  causes  gonorrhea  there  is  no  room 
to  doubt.  It  is  constantly  present  in  the  disease,  and 
very  frequently  also  in  the  sequelae — endometritis,  salpin- 
gitis,  oophoritis,  cystitis,  peritonitis,  arthritis,  conjuncti- 
vitis, etc. — and,  so  far  as  can  at  present  be  determined,  is 
never  found  under  normal  conditions. 

In  the  beginning  of  their  activities  the  cocci  grow  in 
the  superficial  epithelial  cells,  but  soon  penetrate  between 
the  cells  to  the  deeper  layers,  where  they  continue  their 
irritation  as  the  superficial  cells  desquamate.  Authorities 
differ  as  to  whether  the  gonococci  can  penetrate  squamous 
and  columnar  epithelium  with  equal  facility. 

The  peri ure thral  abscesses  that  occur  in  the  course  of 
gonorrhea  are  generally  due  to  the  Staphylococci  aureus 
and  albus,  not  directly  to  the  gonococcus. 

As  long  as  the  gonococci  persist  the  patient  may  spread 
contagion.  It  must  be  pointed  out  that  after  apparent 
recovery  from  the  disease  the  cocci  sometimes  remain 
latent  in  the  urethra,  and  set  up  a  relapse  if  the  patient 
partake  of  some  substance,  as  alcohol,  irritating  to  the 
mucous  membranes.  Bearing  this  in  mind,  patients 
should  not  too  soon  be  discharged  as  cured. 

The  gonococci  are  not  easily  killed,  but  withstand  dry- 


i68  PATHOGENIC  BACTERIA. 

ing  very  well.  Kratter  was  able  to  demonstrate  their 
presence  upon  washed  clothing  six  months  after  the  orig- 
inal soiling,  and  also  found  that  they  still  stained  well. 

Bumm  found  cocci  similar  to  the  gonococcus  in  the 
urethra,  and  points  out  that  the  shape  is  not  characteristic, 
that  the  position  in  the  cells  is  not  positively  diagnostic, 
but  that  added  to  these  characteristics  we  must  have  the 
refusal  to  stain  by  Gram's  method  before  we  can  say  posi- 
tively that  cocci  found  in  urethral  pus  are  gonococci. 


II.    THE   CHRONIC   INFLAMMATORY   DISEASES. 


CHAPTER    I. 
TUBERCULOSIS. 

TUBERCULOSIS  is  one  of  the  most  dreadful  and,  un- 
fortunately, most  common  diseases  of  mankind.  It  affects 
alike  the  young  and  the  old,  the  rich  and  the  poor,  the 
male  and  the  female,  the  enlightened  and  the  savage. 
Nor  do  its  ravages  cease  with  human  beings,  for  it  is 
common  among  animals,  occurring  with  great  frequency 
among  cattle,  less  frequently  among  goats  and  hogs,  and 
sometimes,  though  rarely,  among  sheep,  horses,  dogs, 
and  cats. 

Wild  animals  under  natural  conditions  seem  to  escape 
the  disease,  but  when  caged  and  kept  in  zoological  gar- 
dens even  the  most  resistant  of  them — lions,  tigers,  etc. — 
are  said  at  times  to  succumb  to  it,  while  it  is  the  most 
common  cause  of  death  among  captive  monkeys. 

The  disease  is  not  even  limited  to  mammals,  but  occurs 
in  a  somewhat  modified  form  in  birds,  and,  it  is  said, 
even  at  times  affects  reptiles. 

It  is  not  a  disease  of  modern  times,  but  one  which  has 
persisted  through  centuries ;  and  though,  before  the  ad- 
vent of  the  microscope,  not  always  clearly  separated 
from  cancer,  it  has  not  only  left  unmistakable  signs  of 
its  existence  in  the  early  literature  of  medicine,  but  has 
also  imprinted  itself  upon  the  statute-books  of  some 
countries,  as  Naples,  where  its  ravages  were  great  and 
the  means  taken  for  its  prevention  radical. 

While  the  great  men  of  the  early  days  of  pathology 
clearly  saw  that  the  time  must  come  when  the  parasitic 

169 


170  PATHOGENIC  BACTERIA. 

nature  of  this  disease  would  be  proved,  and  some,  as 
Klebs,  Villemin,  and  Cohnheim,  were  "within  an  ace" 
of  the  discovery,  it  remained  for  Robert  Koch  to  succeed 
in  demonstrating  and  isolating  the  specific  bacillus,  now 
so  well  known,  and  to  write  so  accurate  a  description  of 
the  organism  and  the  lesions  it  produces  as  to  render  it 
almost  unparalleled  in  medical  literature. 

The  tubercle  bacillus  (Fig,  54)  is  a  rod-shaped  organ- 


FIG.  54. — Section  of  a  peritoneal  tubercle  from  a  cow,  showing  the  tubercle 
bacilli;    x  500  (Frankel  and  Pfeiffer). 


ism  with  rounded  ends  and  a  slight  curve,  measuring 
from  1.5-3.5  p  in  length  and  from  0.2-0.5  //  in  breadth. 
It  very  commonly  occurs  in  pairs,  which  may  be  asso- 
ciated end  to  end,  but  generally  overlap  somewhat  and 
are  not  attached  to  each  other.  It  is  very  common  to 
observe  a  peculiar  beaded  appearance  in  organisms  found 
in  pus  and  sputum  (Fig.  55),  due  to  the  contraction  of 
fragmented  protoplasm  within  the  resisting  capsule  (?). 
By  some  these  fragmentations  are  thought  to  be  bacilli 
in  the  stage  of  sporulation.  Koch  originally  held  this 
view  himself,  but  researches  have  not  been  able  to  sub- 
stantiate the  opinion,  and  at  present  the  evidences  pro 


TUBERCULOSIS.  171 

and  con.  point  more  strongly  in  the  negative  than  in  the 
positive  direction. 

The  fragments  do  not  look  like  the  spores  of  any  other 
organisms.  When  spores  occur  in  the  continuity  of 
bacilli,  they  are  generally  discrete  oval  refracting  bodies 
easily  recognized.  The  fragments  seen  in  the  tubercle 
bacillus  are  irregular  and  biconcave  instead  of  oval,  have 

..•  *#&* 

dF5^& 

K##vW«    „-> 


FIG.  55. — Tubercle  bacillus  in  sputum  (Frankel  and  Pfeiffer). 

ragged  surfaces,  and  are  without  the  refraction  peculiar 
to  the  ordinary  spore. 

The  spaces  between  the  bacillary  fragments  cannot  be 
made  to  stain  like  the  spores  of  other  species.  Finally, 
all  known  spores  resist  heat  more  strongly  than  the  fully- 
developed  bacilli,  but  experimentation  has  shown  that 
these  degenerative  forms  are  no  more  capable  of  resist- 
ing heat  than  the  tubercle  bacilli  themselves. 

The  organism  is  not  motile,  and  does  not  possess 
flagella. 

The  tubercle  bacillus  is  peculiar  in  its  reaction  to  the 
anilin  dyes.  It  is  rather  difficult  to  stain,  requiring  that 
the  dye  used  shall  contain  a  mordant  (Koch),  but  it  is  also 
very  tenacious  of  the  color  once  assumed,  resisting  the 
decolorizing  power  of  strong  mineral  acids  (Ehrlich). 


172  PATHOGENIC  BACTERIA. 

These  peculiarities  delayed  the  discovery  of  the  bacil- 
lus for  a  considerable  time,  but  now  that  we  are  familiar 
with  them  they  give  us  a  most  valuable  diagnostic  cha- 
racter, for  with  the  exception  of  the  bacillus  of  lepra  no 
known  bacillus  reacts  in  exactly  the  same  way. 

Koch  first  stained  the  bacillus  with  an  aqueous  solu- 
tion of  a  basic  anilin  dye  to  which  some  potassium 
hydrate  was  added,  subsequently  washing  with  water 
and  counter-staining  with  vesuvin.  Ehrlich  subsequently 
modified  Koch's  method,  showing  that  pure  anilin  was 
a  better  mordant  than  potassium  hydrate,  and  that  the 
use  of  a  strong  mineral  acid  would  remove  the  color 
from  everything  but  the  tubercle  bacillus.  This  modi- 
fication of  Koch's  method  given  us  by  Ehrlich  is  at  the 
present  time  acknowledged  to  be  the  best  method  of 
staining  the  bacillus.  Many  other  methods  have  been 
suggested,  all  of  them,  perhaps,  more  convenient  than 
Ehrlich' s,  but  none  so  good. 

As  being  that  most  frequently  performed  by  the 
physician,  we  will  first  describe  the  method  of  seeking 
the  bacillus  in  sputum. 

If  one  desires  to  be  very  exact  in  his  examination, 
it  may  be  well  to  have  the  patient  cleanse  the  mouth 
thoroughly  upon  waking  in  the  morning,  and  after  the 
first  fit  of  coughing  expectorate  into  a  clean  wide- 
mouthed  bottle.  The  object  of  this  is  to  avoid  the 
presence  of  fragments  of  food  in  the  sputum. 

The  physician  will  secure  a  better  result  if  the  exam- 
ination be  made  on  the  same  day  than  if  he  wait  a  num- 
ber of  days,  because  if  the  bacilli  are  few  they  occur 
most  plentifully  in  the  small  caseous  flakes  to  be  de- 
scribed farther  on,  which  are  easily  found  at  first,  but 
which  break  up  and  become  part  of  a  granular  sediment 
that  always  forms  in  decomposed  sputum. 

The  fresh  sputum  when  held  over  a  black  surface 
generally  shows  a  number  of  grayish-yellow,  irregular, 
translucent  granules  somewhat  smaller  than  the  head  of 
a  pin.  These  consist  principally  of  the  caseous  material 


TUBERCULOSIS.  173 

from  tuberculous  tissue,  and  are  the  most  valuable  part 
of  the  sputum  for  examination.  One  of  the  granules  is 
picked  up  with  a  pointed  match-stick  and  spread  over 
the  surface  of  a  perfectly  clean  cover-glass.  If  no  such 
fragment  can  be  found,  the  purulent  part  is  next  best  for 
examination.  The  mucus  itself  rarely  contains  bacilli 
when  free  from  scraps  of  tissue  and  pus. 

In  cases  in  which  this  ordinary  procedure  fails  to  reveal 
bacilli  whose  presence  is  strongly  indicated  by  the  clin- 
ical signs,  the  exact  method  of  searching  for  them  is  to 
partially  digest  the  sputum  with  caustic  potash,  and  then 
collect  the  solid  matter  with  a  centrifugal  apparatus.  If 
a  very  few  bacilli  are  present  in  the  sputum,  this  method 
will  often  secure  them. 

The  material  spread  upon  the  cover-glasses  should  not 
be  too  small  in  amount.  Of  course  a  massive,  thick 
layer  will  become  opaque  in  staining,  but  should  the 
layer  spread  be,  as  is  often  advised,  "as  thin  as  possible," 
there  may  be  too  few  bacilli  upon  the  glass  to  enable  one 
to  make  a  satisfactory  diagnosis. 

As  usual,  the  material  is  allowed  to  dry  thoroughly, 
and  is  then  passed  three  times  through  the  flame  for 
purposes  of  fixation. 

Ehrlictts  Method,  or  the  Koch-Ehrlich  Method.—  The 
cover-glasses  thus  prepared  are  floated,  smeared  side 
down,  upon,  or  immersed,  smeared  side  up,  in,  a  small 
dish  of  Ehrlich's  anilin-water  gentian- violet  solution  : 

Anilin,  4, 

Saturated  alcoholic  solution  of  gentian  violet,     1 1 , 
Water,  100, 

and  placed  in  an  incubator  or  a  paraffin  oven,  and  kept 
for  twenty-four  hours  at  about  the  temperature  of  the 
body.  When  removed  from  the  stain  they  are  washed 
momentarily  in  water,  and  then  alternately  in  25-33 
per  cent,  nitric  acid  and  60  per  cent,  alcohol,  until  the 
blue  color  of  the  gentian  violet  is  almost  entirely  lost. 
It  must  be  remembered  that  the  action  of  the  strong  acid 


1/4  PATHOGENIC  BACTERIA. 

is  a  powerful  one,  and  that  too  long  a  time  must  not  be 
allowed  for  its  application.  A  total  immersion  of  thirty 
seconds  is  quite  enough  in  most  cases.  After  final  thor- 
ough washing  in  60  per  cent,  alcohol  the  specimen  is 
counter-stained  in  a  dilute  aqueous  solution  of  Bismarck 
brown  or  vesuvin.  The  excess  of  stain  is  then  washed 
off  in  water,  and  the  specimen  is  dried  and  mounted  in 
balsam.  The  tubercle  bacilli  will  appear  of  a  fine  dark 
blue,  while  the  pus-corpuscles,  epithelial  cells,  and  other 
bacteria,  having  been  decolorized  by  the  acid,  will  be 
colored  brown  by  the  counter-stain. 

This  method,  requiring  twenty-four  hours  for  its  com- 
pletion, is  naturally  one  which  has  fallen  into  disuse  for 
practitioners  who  desire  in  the  briefest  possible  time  to 
know  simply  whether  bacilli  are  present  in  the  sputum 
or  not. 

Among  clinicians  Ziehl's  method  with  carbol-fuchsin 
has  met  with  great  favor.  After  having  been  spread, 
dried,  and  fired,  the  cover-glass  is  held  in  the  bite  of  an 
appropriate  forceps  (cover-glass  forceps),  and  the  stain l 
dropped  upon  it  from  a  pipette.  As  soon  as  the  entire 
cover-glass  is  covered  with  stain  it  is  held  over  the  flame 
of  a  spirit-lamp  or  a  Bunsen  burner  until  the  stain  begins 
to  volatilize  a  little,  as  indicated  by  a  white  vapor.  When 
this  is  observed,  the  heating  is  sufficient,  and  the  temper- 
ature can  be  subsequently  maintained  by  intermittent 
heating. 

If  evaporation  is  allowed  to  take  place,  a  ring  of  in- 
crustation occurs  at  the  edge  of  the  area  covered  by  the 
stain  and  prevents  the  proper  action  of  the  acid.  To 
prevent  this  more  stain  should  now  and  then  be  added. 
The  staining  is  complete  in  from  three  to  five  minutes, 
after  which  the  specimen  is  washed  off  with  water,  the 
excess  of  water  absorbed  with  paper,  and  25  per  cent. 

1  Carbol-fuchsin  (see  p.  86) : 

Fuchsin,  I ; 

Alcohol,  10 ; 

5  per  cent,  phenol  in  water.  100. 


OF 


TUBERCULOSIS,  175 

sulphuric  or  33  per  cent,  nitric  acid  dropped  upon  it  for 
thirty  seconds.  The  acid  is  washed  off  with  water,  and 
the  specimen  is  dried  and  mounted  in  Canada  balsam. 
Nothing  will  be  colored  except  the  tubercle  bacilli, 
which  will  appear  red. 

Gabbett  modified  the  staining  by  adding  methylene 
blue  to  the  acid  solution,  which  he  makes  according  to 
this  formula: 

Methyl  blue,  2  ; 

Sulphuric  acid,  25  ; 

Water,  75. 

In  Gabbett' s  method,  after  staining  with  carbol-fuch- 
sin  the  specimen  is  washed  with  water,  acted  upon  by 
the  methylene-blue  solution  for  exactly  thirty  seconds, 
washed  with  water  until  only  a  very  faint  blue  remains, 
dried,  and  finally  mounted  in  Canada  balsam.  By  this 
method  the  tubercle  bacilli  are  colored  red,  and  the  pus- 
corpuscles,  epithelial  cells,  and  the  unimportant  bacteria 
blue. 

When  the  tubercle  bacilli  are  to  be  sought  for  in  sections 
of  tissue,  considerable  difficulty  is  at  once  encountered, 
partly  because  of  the  thickness  of  the  section  and  partly 
because  of  the  presence  of  nuclei  which  color  intensely. 

Again,  Khrlich's  method  must  be  recommended  as  the 
most  certain  and  best  method  of  staining  a  large  number 
of  bacilli. 

The  sections  of  tissue,  if  imbedded  in  celloidin  or  par- 
affin, should  be  freed  from  the  foreign  substances.  Like 
the  cover-glasses,  they  are  placed  in  the  stain  for  twelve 
to  twenty-four  hours  at  a  temperature  of  37°  C.  Upon 
removal  they  are  allowed  to  lie  in  water  for  about  ten 
minutes  to  wash  away  the  excess  of  stain  and  to  soften 
the  tissue,  which  often  shrinks  and  becomes  brittle.  The 
washing  in  nitric  acid  (20  per  cent.)  which  follows  may 
have  to  be  continued  for  as  long  as  two  minutes.  Thor- 
ough washing  in  60  per  cent,  alcohol  follows,  after  which 
the  sections  can  be  counter-stained,  washed,  dehydrated 


176  PATHOGENIC  BACTERIA. 

in  95  per  cent,  and  absolute  alcohol,  cleared  in  xylol, 
and  mounted  in  Canada  balsam. 

A  method  which  has  attained  great  and  deserved  praise 
is  Unna's.  It  is  as  follows:  The  sections  are  placed  in 
a  dish  of  twenty- four-hours-old,  newly-filtered  Ehrlich's 
solution,  and  allowed  to  remain  twelve  to  twenty-four 
hours  at  the  room-temperature  or  one  to  two  hours  in 
the  incubator.  From  the  stain  they  are  placed  in  water, 
where  they  remain  for  about  ten  minutes  to  wash.  They 
are  next  immersed  in  acid  (20  per  cent,  nitric  acid)  for 
about  two  minutes,  and  become  greenish-black.  From 
the  acid  they  are  placed  in  absolute  alcohol,  and  are 
gently  moved  to  and  fro  until  the  pale-blue  color  returns. 
They  are  then  washed  in  three  or  four  changes  of  clean 
water  until  they  become  almost  colorless,  and  are  then 
removed  to  the  slide  by  means  of  a  section-lifter.  The 
water  is  absorbed  with  filter-paper,  and  then  the  slide  is 
heated  over  a  Bunsen  burner  until  the  section  becomes 
shining,  when  it  receives  a  drop  of  xylol  balsam  and  a 
cover-glass. 

It  is  said  that  sections  stained  in  this  manner  do  not 
fade  as  quickly  as  those  stained  by  Ehrlich's  method. 

The  tubercle  bacillus  also  stains  well  by  Gram's  method, 
but  as  this  is  a  general  method  by  which  many  different 
bacteria  are  colored,  it  is  ill  adapted  for  purposes  of  differ- 
entiation, especially  when  the  prosecution  of  the  charac- 
teristic methods  is  not  more  difficult. 

So  far  as  is  known,  the  tubercle  bacillus  is  a  purely 
parasitic  organism.  It  has  never  been  found  except  in 
the  bodies  and  excretions  of  animals  affected  with  tuber- 
culosis, and  in  dusts  of  which  these  are  component  parts. 
This  purely  parasitic  nature  greatly  interferes  with  the 
isolation  of  the  organism,  which  cannot  be  grown  upon 
the  ordinary  culture-media.  Koch  first  achieved  its  arti- 
ficial cultivation  by  the  use  of  blood-serum.  When 
planted  upon  this  medium  the  bacilli  are  first  apparent 
to  the  naked  eye  in  about  two  weeks,  and  occur  in  the 
form  of  small  dry,  whitish  flakes,  not  unlike  fragments 


TUBERCULOSIS. 


177 


of  chalk.  These  slowly  increase  at  the  edges,  and  grad- 
ually form  scale-like  masses  of  small  size,  which  under 
the  microscope  are  seen  to  consist  of  tangled  masses  of 
bacilli,  many  of  which  are  in  a  condition  of  involution. 


FIG.  56. — Bacillus  tuberculosis  :  adhesive  cover-glass  preparation  from  a  fourteen- 
day-old  blood-serum  culture;    x   100  (Frankel  and  Pfeiffer). 

The  best  method  of  obtaining  a  culture  is  to  inoculate 
a  guinea-pig  with  tuberculous  material,  allow  an  artificial 
tuberculosis  to  develop,  kill  the  animal  after  a  couple  of 
months,  and  make  the  cultures  from  the  centre  of  one  of 
the  tuberculous  glands. 

Of  course  many  technical  difficulties  must  be  over- 
come. The  tuberculous  material  used  for  inoculation 
may  be  sputum,  injected  beneath  the  skin  by  a  hypo- 
dermic syringe.  The  animal  is  allowed  to  live  for  a 
month  or  six  weeks,  then  killed.  The  autopsy  is  per- 
formed according  to  directions  already  given.  A  large 
lymphatic  gland  with  softened  contents  or  a  nodule  in  the 
spleen  being  selected  for  the  culture,  an  incision  is  made 
into  it  with  a  sterile  knife,  or  a  rigid  sterile  platinum 
wire  is  introduced ;  some  of  the  contents  are  removed 
and  planted  upon  blood-serum.  After  receiving  the  in- 
oculated material  the  tubes  are  closed,  either  by  a  rub- 
12 


178  PATHOGENIC  BACTERIA. 

her  cap  placed  over  the  cotton  stopper,  which  is  cut  off 
and  pushed  in,  or  by  a  rubber  cork  above  the  cotton, 
the  idea  of  this  rubber  corking  being  simply  to  prevent 
evaporation.  The  tubes  must  be  kept  in  an  incubator 
at  the  temperature  of  37-38°  C. 

Kitasato  has  published  a  method  by  which  Koch  has 
been  able  to  secure  the  tubercle  bacillus  in  pure  culture 
from  sputum.  After  carefully  cleansing  the  mouth  the 
patient  is  allowed  to  expectorate  into  a  sterile  Petri  dish. 
By  this  method  the  contaminating  bacteria  from  the 
mouth  and  the  receptacle  are  excluded,  and  the  expecto- 
rated material  is  made  to  contain  only  such  bacteria  as 
were  present  in  the  lungs.  The  material  is  carefully 
washed  a  great  many  times  in  renewed  distilled  sterile 
water  until  all  bacteria  not  enclosed  in  the  muco-purulent 
material  are  removed  ;  it  is  then  carefully  opened  with 
sterile  instruments,  and  the  culture-medium — glycerin 
agar-agar  or  blood-serum — is  inoculated  from  the  centre. 
Kitasato  has  been  able  by  this  method  to  demonstrate 
that  many  of  the  bacilli  ordinarily  present  in  tubercular 
sputum  are  dead,  although  they  continue  to  stain  well. 

In  1887,  Nocard  and  Roux  gave  a  great  impetus  to 
investigations  upon  tuberculosis  by  their  discovery  that 
the  addition  of  4-8  per  cent,  of  glycerin  to  bouillon  and 
agar-agar  would  make  them  suitable  for  the  development 
of  the  bacillus,  and  that  a  much  more  luxuriant  develop- 
ment could  be  obtained  upon  these  media  than  upon 
blood-serum.  The  growth  upon  such  "glycerin  agar- 
agar"  much  resembles  that  upon  blood-serum  (Fig.  56). 
The  growth  upon  bouillon  with  4  per  cent,  of  glycerin 
is  also  luxuriant.  As  tubercle  bacilli  require  considerable 
oxygen  for  their  proper  development,  they  grow  only 
upon  the  surface  of  the  bouillon,  where  a  rather  thick 
mycoderma  forms.  The  surface-growth  is  rather  brittle, 
and  after  a  time  gradually  subsides  fragment  by  fragment. 

The  tubercle  bacillus  can  be  grown  in  gelatin  to  which 
glycerin  is  added,  but  as  its  development  only  takes  place 
at  37-38°  C.,  a  temperature  at  which  gelatin  is  always 


TUBERCULOSIS.  179 

liquid,  its  use  for  the  purpose  is  disadvantageous  rather 
than  useful. 

Pawlowski  was  able  to  cultivate  the  bacillus  upon 
potato,  but  Sander,  who  found  that  it  could  be  readily 
grown  upon  various  vegetable  compounds,  especially 
upon  acid  potato  mixed  with  glycerin,  also  found  that 
upon  such  compounds  its  virulence  was  constantly  lost. 

It  has  also  been  shown  that  the  continued  cultivation 
of  the  tubercle  bacillus  upon  such  culture-media  as 
are  appropriate  so  lessens  its  parasitic  nature  that  in  the 
course  of  time  it  can  be  induced  to  grow  feebly  upon  the 
ordinary  agar-agar. 

It  is  really  surprising  to  note  the  extremely  simple 
compounds  in  which  the  tubercle  bacillus  can  be  grown. 
Instead  of  requiring  the  most  concentrated  albuminous 
media,  as  was  once  supposed,  Proskauer  and  Beck  have 
shown  that  the  organism  can  grow  in  non-albuminous 
media  containing  asparagin,  and  that  it  can  even  be  in- 
duced to  grow  upon  a  mixture  of  commercial  ammonium 
carbonate,  0.35  per  cent.;  primary  potassium  phosphate, 
0.15  per  cent;  magnesium  sulphate,  0.25  per  cent.; 
glycerin,  1.5  per  cent.  It  was  even  found  that  tuberculin 
was  produced  in  this  inorganic  mixture. 

The  tubercle  bacillus  seems  to  require  a  considerable 
amount  of  oxygen  for  its  development.  It  is  also  pecu- 
liarly sensitive  to  temperatures,  not  growing  at  a  tem- 
perature below  29°  C.  or  above  42°  C.  Temperatures 
above  75°  C.  kill  it  after  a  short  exposure. 

The  tubercle  bacillus  does  not  develop  well  in  the 
light,  and  when  its  virulence  is  to  be  maintained  should 
always  be  kept  in  the  dark.  Sunlight  kills  it  in  from 
a  few  minutes  to  several  hours,  according  to  the  thick- 
ness of  the  mass  exposed  to  its  influence. 

The  widespread  character  of  tuberculosis  at  one  time 
suggested  the  idea  that  tubercle  bacilli  were  ubiquitous 
in  the  atmosphere,  that  we  all  inhaled  them,  and  that  it 
was  only  our  vital  resistance  that  prevented  us  all  from 
becoming  its  victims. 


i8o  PA  THOGENIC  BA CTERIA . 

Cornet  must  be  given  the  credit  of  having  shown  that 
such  an  idea  is  untrue,  and  that  tubercle  bacilli  only 
exist  in  the  atmospheres  frequented  by  consumptives. 
His  experiments  were  made  by  collecting  dusts  from 
numerous  places — streets,  sidewalks,  houses,  rooms,  walls, 
etc.  Injecting  them  into  guinea-pigs,  whose  constant 
susceptibility  to  the  disease  makes  them  a  very  delicate 
reagent  for  its  detection,  Cornet  showed  the  bacilli  to  be 
present  only  in  the  dust  with  which  pulverized  sputum 
was  mixed,  and  found  such  infectious  dust  to  be  most 
common  where  the  greatest  carelessness  in  respect  to 
cleanliness  prevailed. 

Our  present  knowledge  of  the  life-history  of  the  tubercle 
bacillus,  by  showing  its  indisposition  to  multiply  outside 
the  bodies  of  animals,  the  deleterious  influence  of  sun- 
light upon  it,  the  absence  of  positive  permanent  forms, 
and  its  sensitivity  to  temperatures  beyond  a  certain  range, 
confirms  all  that  Cornet  has  pointed  out,  and  shows  us 
why  the  expectoration  of  millions  of  consumptives  has 
not  rendered  our  atmospheres  pestilential. 

As  long  as  tuberculosis  exists  among  men  or  cattle,  it 
shows  that  the  existing  hygienic  precautions  are  insuf- 
ficient. While  not  so  radical  as  to  suggest  the  unreason- 
able isolation  of  patients  and  destruction  of  property  once 
practised  in  the  kingdom  of  Naples,  the  author  would 
favor  the  registration  of  all  tuberculous  cases  as  a  means 
of  collecting  accurate  data  concerning  their  origin,  would 
insist  upon  domestic  sterilization  and  disinfection,  and 
would  have  special  hospitals  for  as  many,  especially  of 
the  poorer  classes,  among  whom  hygienic  measures  are 
almost  always  opposed,  as  could  be  persuaded  to  occupy 
them. 

It  has  already  been  declared  the  duty  of  the  physician 
to  use  every  means  in  his  power  to  prevent  the  spread 
of  infection  in  the  households  in  his  care,  and  no  disease 
is  more  deserving  of  attention  than  this  neglected  one. 
Patients  should  cease  to  kiss  the  members  of  their  fam- 


TUBERCULOSIS.  181 

ily  and  friends ;  their  individual  knives,  forks,  spoons, 
cups,  etc.  should  be  carefully  kept  apart — secretly  if  the 
patient  be  sensitive  upon  the  subject — from  those  of  the 
family,  and  scalded  after  each  meal ;  the  napkins  and 
handkerchiefs,  as  well  as  whatever  clothing  or  bed-cloth- 
ing is  soiled  by  the  discharges,  should  be  kept  apart  from 
the  common  wash,  and  boiled ;  and  of  course  the  expec- 
toration should  be  carefully  attended  to,  received  in  a 
suitable  receptacle,  sterilized  or  disinfected,  and  never 
allowed  to  dry,  for  it  has  been  shown  that  the  tubercle 
bacillus  can  remain  vital  in  dried  sputum  for  as  long  as 
nine  months.  A  very  neat  arrangement  for  collecting 
and  disposing  of  the  expectoration  is  recommended  by 
some  boards  of  health.  It  consists  of  a  metal  case  into 
which  a  pasteboard  box  is  fitted.  When  the  box  is  to  be 
emptied  the  whole  of  the  pasteboard  portion  is  removed, 
and,  together  with  the  expectoration,  burned.  The  metal 
part  is  disinfected,  provided  with  a  new  pasteboard  box, 
and  is  again  ready  for  use.  (See  Fig.  16,  page  102.)  The 
physician  should  also  give  directions  for  disinfecting  the 
bedroom  occupied  by  a  consumptive  before  it  becomes 
the  chamber  of  a  healthy  person. 

Boards  of  health  are  now  becoming  more  and  more  in- 
terested in  tuberculosis,  and,  though  exceedingly  slow 
and  conservative  in  their  movements,  are  disseminating 
literature  among  doctors  for  distribution  to  their  patients, 
with  the  hope  of  achieving  by  volition  that  which  they 
would  otherwise  regard  as  cruel  compulsion. 

The  channels  by  which  the  tubercle  bacillus  enters  the 
organism  are  varied.  A  few  cases  are  on  record  where 
the  micro-organisms  have  passed  through  the  placenta, 
so  that  a  tuberculous  mother  was  able  to  infect  her 
unborn  child.  It  is  not  impossible  that  the  passage  of 
bacilli  in  this  manner  through  the  placenta  causes  the 
development  of  tuberculosis  in  infants  after  birth,  the 
disease  having  remained  latent  during  fetal  life,  for 
Birch-Hirschfeld  has  shown  that  fragments  of  a  fetus, 
itself  showing  no  tubercular  lesions,  but  coming  from  a 


i83  PA  THOGENIC  BACTERIA . 

tuberculous  woman,  were  fatal  to  guinea-pigs  into  which 
they  were  inoculated. 

The  most  frequent  channel  of  infection  is  the  respira- 
tory tract,  into  which  the  finely-pulverized  dust  of  rooms 
and  streets  enters.  Probably  all  of  us  at  some  time  in 
our  lives  inhale  living  virulent  tubercle  bacilli,  yet  not 
all  of  us  suffer  from  tuberculosis.  Personal  predisposi- 
tion seems  of  great  importance,  for  it  has  been  shown 
that  without  the  formation  of  tubercles  virulent  bacilli 
may  be  present  for  considerable  lengths  of  time  in  the 
bronchial  lymphatic  glands — the  dumping-ground  of  the 
pulmonary  phagocytes. 

In  order  that  infection  shall  occur  it  does  not  seem 
necessary  that  the  least  abrasion  or  laceration  shall  exist 
in  the  mucous  lining  of  the  respiratory  tract.  The 
tubercle  bacillus  is  a  foreign  body  of  irritating  prop- 
erties, and,  lodging  upon  a  cell,  is  soon  engulfed  in  its 
protoplasm,  or,  arrested  by  a  leucocyte,  is  dragged  off  to 
some  other  region  in  whose  narrow  passages  a  most  hos- 
tile strife  doubtless  takes  place. 

Infection  also  commonly  takes  place  through  the  g as- 
tro-intestinal tract  by  infected  food.  At  present  an  over- 
whelming weight  of  evidence  points  to  the  presence  of 
bacilli  in  the  milk  of  cattle  affected  with  tuberculosis.  It 
does  not  seem  necessary  that  tuberculous  ulcers  shall  be 
present  in  the  udders ;  indeed,  the  bacilli  have  been 
demonstrated  in  considerable  numbers  in  milk  from 
udders  without  tubercular  lesions  discoverable  to  the 
naked  eye. 

The  meat  from  tuberculous  animals  is  less  dangerous 
than  the  milk,  because  the  meat  is  nearly  always  cooked 
before  being  eaten,  while  the  milk  is  generally  taken 
uncooked.  The  bacilli  enter  the  intestinal  lymphatics, 
sometimes  produce  lesions  immediately  beneath  the  mu- 
cous membrane,  and  lead  later  on  to  the  formation  of 
ulcers  ;  but  generally  they  first  involve  the  mesenteric 
lymphatic  glands.  The  thoracic,  duct  is  sometimes  af- 
fected, and  from  such  a  lesion  it  is  easy  to  understand  the 


TUBERCULOSIS.  183 

development  of  a  general  miliary  tuberculosis.  The  oc- 
casional absorption  of  tubercle  bacilli  by  the  lacteals,  and 
their  entrance  into  the  systemic  circulation  and  subse- 
quent deposition  in  the  brain,  bones,  joints,  etc.,  are  sup- 
posed to  explain  primary  lesions  of  these  tissues. 

Infection  is  said  also  to  take  place  occasionally  through 
the  sexual  apparatus.  In  sexual  intercourse  tubercle 
bacilli  from  tuberculous  testicles  may  be  discharged  into 
the  female  organs,  with  resulting  tuberculous  lesions. 
The  infection  in  this  way  generally  is  from  the  male  to 
the  female,  primary  tuberculosis  of  the  testicle  being 
much  more  common  than  primary  tuberculosis  of  the 
uterus  or  ovaries. 

While  most  probably  rare,  in  comparison  with  the 
preceding,  'wounds  also  are  avenues  of  entrance  for  the 
tubercle  bacilli.  Anatomical  tubercles  are  not  uncom- 
mon upon  the  hands  of  anatomists  and  pathologists, 
most  of  these  growths  being  tuberculous  in  character. 
An  interesting  fact  concerning  these  dermal  lesions 
is  the  exceedingly  small  number  of  bacilli  which  they 
contain. 

The  macroscopic  lesions  of  tuberculosis  are  too  familiar 
to  require  a  description  of  any  considerable  length.  They 
consist  in  nodes,  nodules,  or  collections  of  agminated 
nodules,  called  tubercles,  scattered  irregularly  through 
the  tissues,  which  are  devitalized  or  disorganized  by 
their  presence.  When  tubercle  bacilli  are  introduced 
beneath  the  skin  of  a  guinea-pig,  the  animal  shows  no 
sign  of  disease  for  a  week  or  two  ;  it  then  begins  to  lose 
appetite  and  gradually  to  diminish  in  flesh  and  weight. 
Examination  at  this  time  will  show  a  nodule  at  the  point 
of  injection  and  enlargement  of  the  neighboring  lymphatic 
glands.  The  atrophy  increases,  the  animal  shows  a  febrile 
reaction,  and  at  the  end  of  a  varying  period  of  time, 
averaging  about  twelve  weeks,  dies.  Post-mortem  ex- 
amination shows  a  cluster  of  tubercles  at  the  point  of 
inoculation,  enlargement  of  lymphatic  glands  both  near 
and  remote  from  the  primary  lesion  (due  to  the  presence 


1 84  PATHOGENIC  BACTERIA. 

of  tubercles),  and  a  widespread  invasion  of  the  lungs, 
liver,  kidneys,  peritoneum,  and  other  organs  and  tissues, 
with  tuberculous  tissue  in  a  more  or  less  advanced  con- 
dition of  necrosis.  Sometimes  there  are  no  tubercles 
discoverable  at  the  point  of  inoculation.  There  is  no 
regularity  in  the  distribution  of  the  disease.  Tubercle 
bacilli  are  demonstrable  in  immense  numbers  in  all  the 
diseased  tissues.  The  disease  as  seen  in  the  guinea-pig  is 
more  extended  than  in  other  animals  because  of  its  greater 
susceptibility,  and  the  death  of  the  animal  is  more  rapid 
than  in  other  species  for  the  same  reason.  In  rabbits  the 
lesion  runs  a  longer  course  with  similar  lesions.  In 
bovines  and  sheep  the  infection  is  generally  first  seen 
in,  and  is  principally  confined  to,  the  alimentary  appa- 
ratus and  the  associated  organs,  though  pulmonary  dis- 
ease also  occurs.  In  man  the  disease  is  chiefly  pulmonary, 
though  gastro-intestinal  and  general  miliary  forms  are  also 
common.  The  development  of  the  lesions  in  whatever 
tissue  or  animal  always  depends  upon  the  distribution  of 
the  bacilli  by  the  lymph  or  the  blood,  and  is  first  inflam- 
matory, then  degenerative,  in  type. 

The  experiments  of  Koch,  Prudden  and  Hodenphyl, 
and  others  have  shown  that  when  dead  tubercle  bacilli 
are  injected  into  the  subcutaneous  tissues  of  rabbits 
small  local  abscesses  develop  in  the  course  of  a  couple 
of  weeks,  showing  that  the  tubercle  bacilli  are  chemotac- 
tically  potent. 

While  it  is  extremely  interesting  to  observe  that  this 
chemotactic  property  exists,  it  seems  to  be  by  some  other 
irritant  that  most  of  the  lesions  of  tuberculosis  are  caused. 
When  the  dead  tubercle  bacilli,  instead  of  being  injected 
en  masse  into  the  areolar  tissue,  are  so  introduced  info 
the  body — as  by  intravenous  injection — as  to  disseminate 
themselves  or  remain  in  small  groups,  the  result  is  quite 
different,  and  much  more  closely  resembles  that  of  the 
action  of  the  living  organism. 

Baumgarten,  whose  researches  were  made  upon  minute 
tubercles  of  the  iris,  has  shown  that  the  first  manifesta- 


TUBERCULOSIS.  185 

tion  of  the  irritation  caused  by  the  bacillus  is  not  the 
attraction  of  leucocytes,  but  the  stimulation  of  the  fixed 
connective-tissue  cells  of  the  part  affected.  These  cells 
increase  in  number  by  karyokinesis,  and  form  about  the 
irritating  bacterium  a  minute  focus  which  is  the  primitive 
tubercle. 

The  leucocytes  are  of  secondary  advent,  and  are  no 
doubt  attracted  both  by  the  substance  shown  by  Prudden 
and  Hodenphyl  to  exist  in  the  bodies  of  the  dead  bacilli 
and  by  the  necrotic  changes  which  already  affect  the 
primary  cells.  For  reasons  not  understood,  the  amount 
of  chemotaxis  varies  greatly  in  different  cases.  Some- 
times the  tubercles  will  be  sufficiently  purulent  in  type 
almost  to  justify  the  name  "tubercular  abscess;"  some- 
times there  will  be  a  marked  absence  of  cellular  ele- 
ments derived  from  the  blood. 

The  important  toxic  substance  produced  by  the  bacillus 
is  evidently  not  associated  with  chemotaxis,  for  when  the 
leucocytes  are  absent  the  necrosis  which  is  so  characteris- 
tic persists. 

The  groups  of  cells  constituting  the  primitive  tubercle 
have  scarcely  reached  microscopic  proportions  before  a 
distinct  coagulation-necrosis  is  observable.  The  proto- 
plasm of  the  cells  affected  takes  on  a  hyaline  character, 
and  seems  abnormally  viscid,  so  that  contiguous  cells 
have  a  tendency  to  become  partially  confluent.  The 
chromatin  of  their  nuclei  becomes  dissolved  in  the  nu- 
clear juice  and  gives  stained  nuclei  a  pale  but  homo- 
geneous appearance.  Sometimes  this  nuclear  change  is 
only  observed  very  late.  As  the  necrosis  advances  the 
contiguous  cells  flow  together  and  form  large  protoplas- 
mic masses — giant-cells — which  contain  as  many  nuclei 
as  there  were  component  cells.  It  may  be  that  these 
nuclei  multiply  by  karyokinesis  after  the  protoplasmic 
coalescence,  but  only  one  observer,  Baumgarten,  has 
found  signs  of  this  process  in  giant-cells.  While  these 
changes  are  in  progress  in  the  cells  of  the  primary  focus, 
the  leucocytes  may  collect  in  such  numbers  as  to  obscure 


186  PATHOGENIC  BACTERIA. 

them  and  make  themselves  appear  to  constitute  the  prim- 
itive cells.  When  the  irritant  substance  is  produced  in 
considerable  quantities,  the  most  delicate  cells  die  first ; 
and  it  is  not  infrequent  to  find  a  tubercle  rich  in  leuco- 
cytes suddenly  showing  degeneration  of  these  cells,  with 
recurring  prominence  of  the  original  epithelioid  cells. 

It  has  been  taught  by  some  that  the  giant-cells  are 
produced  by  the  union  of  the  leucocytes,  but  a  careful 
observation  of  the  role  played  by  these  cells  will  convince 
one  that  such  an  origin  for  these  monstrous  cells  must  be 
very  rare. 

Giant-cells  are  not  always  produced,  for  sometimes  the 
necrotic  changes  are  so  violent  and  widespread  as  to  con- 
vert the  whole  cellular  mass  into  a  granular  detritus  of 
unrecognizable  fragments. 

Tubercles  are  constantly  avascular,  as  would  be  ex- 
pected of  a  process  which  is  a  combination  of  progressive 
irritation  and  necrosis.  The  avascularity  may  be  a  fac- 
tor in  the  necrosis  of  the  larger  tuberculous  masses,  but 
it  plays  no  part  in  the  degeneration  of  the  smallest  tuber- 
cles, which  is  purely  toxic. 

Tubercles  may  be  developed  in  any  tissue  and  in  any 
organ.  In  whatever  situation  they  occur,  space  is  occu- 
pied at  the  expense  of  the  tissue,  whose  component  cells 
are  pushed  aside  or  else  included  in  the  nodule.  In  mil- 
iary  tuberculosis  of  the  kidney  it  is  not  unusual  to  find  a 
tubercle  including  a  whole  glomerule,  and  resolving  its 
component  thrombosed  capillaries  and  epithelium  into 
necrotic  fragments. 

As  almost  all  tissues  contain  a  supporting  tissue-frame- 
work of  connective-tissue  fragments,  some  of  these  must 
be  embodied  in  the  new  growth.  The  fibres  which  pos- 
sess little  vitality  are  more  resistant  than  cells,  and,  after 
all  the  cells  of  a  tubercle  have  been  destroyed,  will  be 
distinctly  visible  among  the  granules,  so  that  the  tubercle 
has  a  reticulated  appearance. 

As  a  rule,  tubercles  steadily  increase  in  size  by  the  in- 
vasion of  fresh  tissue.  The  tubercle  bacillus  does  not  seem 


TUBERCULOSIS.  187 

to  find  the  necrotic  centres  of  the  tubercles  adapted  to  its 
growth,  and  completes  its  life-cycle  with  the  tissue-cells. 
It  is  unusual  to  find  healthy-looking  bacilli  in  the  necrotic 
areas,  most  of  them  being  observed  at  the  edges  of  the 
tubercle,  where  the  nutrition  is  good.  From  such  edges 
the  bacilli  are  occasionally  picked  up  by  leucocytes  and 
transported  through  the  lymph-spaces,  until  the  phago- 
cyte falls  a  prey  to  its  prisoner,  dies,  and  sows  the  seed 
of  a  new  tubercle.  However,  for  the  spread  of  tubercle 
bacilli  from  place  to  place  phagocytes  are  not  always 
necessary,  for  the  bacilli  seem  capable  of  transportation 
by  streams  of  lymph  alone. 

Notwithstanding  the  steady  advance  which  takes  place 
in  most  observed  cases  of  tuberculosis,  and  the  thoroughly 
comprehensible  microscopic  explanation  of  it,  many  cases 
of  tuberculosis  make  quite  perfect  recoveries. 

The  periphery  of  every  tubercle  is  a  zone  of  reaction, 
with  a  marked  tendency  to  granulation  and  organization. 
If  the  vital  condition  is  such  that  through  inappro- 
priate nutriment  or  through  unusually  active  phago- 
cytosis the  activity  of  the  bacilli  is  checked  or  their 
death  is  brought  about,  this  tendency  to  cicatrization  is 
allowed  to  progress  unmolested,  and  the  necrosed  mass  is 
soon  surrounded  with  a  zone  of  newly-formed  contracting 
fibrillar  tissue,  by  which  it  is  perfectly  isolated.  In  such 
isolated  masses  lime-salts  are  commonly  deposited.  Some- 
times this  process  is  perfected  without  the  destruction  of 
the  bacilli,  but  with  their  incarceration  and  inhibition. 
Such  a  condition  is  called  latent  tuberculosis,  and  may  at 
any  time  be  the  starting-point  of  a  new  infection  and  lead 
to  a  fatal  termination. 

In  1890,  Koch  announced  some  observations  upon  toxic 
products  of  the  tubercle  bacillus  and  their  relation  to  the 
diagnosis  and  treatment  of  tuberculosis,  which  at  once 
aroused  an  enormous  but,  unfortunately,  a  transitory 
enthusiasm. 

These  observations,  however,  are  of  capital  importance. 
Koch  observed  that  when  guinea-pigs  are  inoculated 


i88  PATHOGENIC  BACTERIA. 

with  a  mixture  containing  tubercle  bacilli  the  wound 
ordinarily  heals  readily,  and  soon  all  signs  of  local  dis- 
turbance other  than  enlargement  of  the  lymphatic  glands 
of  the  neighborhood  disappear.  In  about  two  weeks  there 
occurs  at  the  point  of  inoculation  a  slight  induration  which 
develops  into  a  hard  nodule,  then  ulcerates,  and  remains 
until  the  death  of  the  animal.  If,  however,  in  the  course 
of  a  short  time  the  animals  are  reinoculated,  the  course 
of  the  process  is  altogether  changed,  for,  instead  of  heal- 
ing, the  wound  and  the  tissue  surrounding  it  assume 
a  dark  color  and  become  obviously  necrotic,  and  ulti- 
mately slough  away,  leaving  an  ulcer  which  rapidly  and 
permanently  heals  without  enlargement  of  the  lymph- 
glands. 

Having  made  this  observation  with  injected  cultures 
of  the  living  bacillus,  Koch  next  observed  that  the  same 
change  occurred  when  the  secondary  inoculation  was 
made  with  pure  cultures  of  the  dead  bacilli. 

It  was  also  observed  that  if  the  material  used  for  the 
secondary  injection  was  not  too  concentrated  and  not 
too  often  repeated  (only  every  six  to  forty-eight  hours), 
the  animals  thus  treated  improved  in  condition,  and, 
instead  of  dying  of  the  tuberculosis  induced  by  the 
primary  injection  in  from  six  to  ten  weeks,  continued 
to  live,  sometimes  (Pfuhl)  as  long  as  nineteen  weeks. 

Koch  also  discovered  that  a  50  per  cent,  glycerin 
extract  of  cultures  of  the  tubercle  bacillus  produced  the 
same  effect  as  the  dead  cultures  originally  used,  and 
gave  this  substance,  tuberculin,  to  the  scientific  world 
for  experimental  purposes,  in  the  hope  that  the  prolon- 
gation of  life  observed  in  the  guinea-pig  might  be  true 
in  the  case  of  man. 

The  active  substance  of  the  u  tuberculin"  seems  to  be 
an  albuminous  derivative  insoluble  in  absolute  alcohol. 
It  is  not  a  toxalbumin. 

The  action  of  the  tuberculin  upon  the  animal  organ- 
ism is  peculiar,  but  readily  understandable.  //  does  not 
exert  the  slightest  influence  upon  the  tubercle  bacillus, 


TUBERCULOSIS.  189 

but  acts  upon  the  living  tuberculous  tissue.  In  the 
description  of  the  tissue-changes  already  given  it  has 
been  shown  that  the  tubercle  bacillus  effects  the  coagu- 
lation-necrosis of  the  cells,  but  does  not  derive  its  nutri- 
ment from  the  dead  tissue.  As  the  cells  die  and  are 
incorporated  in  the  necrotic  mass,  the  bacilli  find  the 
conditions  of  life  unfavorable,  and  likewise  seem  to  die. 
The  active  bacilli,  therefore,  are  always  found  at  the  mar- 
gins of  the  tuberculous  tissues,  where  the  cells  are  fairly 
active.  The  necrosis  is  due  to  bacillary  poisons.  When 
tuberculin  is  injected  into  the  organism  the  result  is  to 
double  the  amount  of  poisonous  influence  upon  the  cells 
surrounding  the  bacilli,  to  destroy  their  vitality,  to  re- 
move the  favorable  conditions  of  growth  from  the  organ- 
ism, and  to  leave  it  for  a  time  checkmated. 

Virchow,  who  well  understood  the  action  of  the  tuber- 
culin, soon  showed  that  as  a  diagnostic  and  therapeutic 
agent  in  man  its  use  was  attended  with  great  danger. 
The  destroyed  tissue  was  absorbed,  and  with  it  the  bacilli 
were  likewise  absorbed  and  transported  to  new  areas, 
where  a  rapid  invasion  occurred.  Old  tuberculous  lesions 
which  had  been  encapsulated  were  softened,  broken 
down,  and  became  sources  of  dangerous  infection  to  the 
individual,  so  that,  a  short  time  after  its  enthusiastic 
reception  as  a  "gift  of  the  gods,"  tuberculin  was  placed 
upon  its  proper  footing  as  a  diagnostic  agent  valuable  in 
veterinary  practice,  but  dangerous  in  human  medicine, 
except  in  cases  of  lupus  and  other  external  forms  of  the 
disease  where  the  destroyed  tissue  could  be  discharged 
from  the  surface  of  the  body. 

The  method  of  preparation  of  tuberculin  is  rather 
simple.  Small  flasks  exposing  a  considerable  surface  of 
liquid  are  filled  with  about  25  c.cm.  of  bouillon  contain- 
ing about  4  per  cent,  of  glycerin.  The  bouillon  is  prefer- 
ably made  with  calf-  instead  of  ox-meat.  When  thor- 
oughly sterile  the  surfaces  are  inoculated  with  pure 
cultures  of  the  tubercle  bacillus  and  are  stood  in  an 
incubator.  In  the  course  of  two  weeks  a  slight  surface 


190  PATHOGENIC  BACTERIA. 

growth  is  apparent,  which  in  the  course  of  time  develops 
into  a  pretty  firm  pellicle  and  gradually  subsides.  At  the 
end  of  four  or  six  weeks  development  ceases  and  the 
pellicle  sinks.  The  contents  of  a  number  of  flasks  are 
then  collected  in  an  appropriate  vessel  and  evaporated 
over  a  water-bath  to  one-tenth  their  volume,  then  filtered 
through  a  Pasteur-Chamberland  filter.  This  is  crude 
tuberculin. 

When  such  a  product  is  injected  in  doses  of  a  fraction 
of  a  cubic  centimeter  an  inflammatory  and  febrile  reac- 
tion occurs.  The  inflammation  sometimes  causes  super- 
ficial tuberculous  lesions  (lupus)  to  ulcerate  and  slough 
away,  and  for  this  reason  is  of  some  value  in  therapeutics, 
although  attended  with  the  dangers  mentioned  above. 
The  fever  is  sufficiently  characteristic  to  be  of  diagnostic 
value,  though  the  tuberculin  can  only  be  used  as  a  diag- 
nostic agent  in  practice  upon  animals. 

Numerous  experimenters,  prominent  among  whom  are 
Tizzoni,  Cattani,  Bernheim,  and  Paquin,  have  experi- 
mented with  the  tubercle  bacillus  and  tuberculin,  hoping 
that  the  principles  of  serum-therapy  might  be  applicable 
to  the  disease.  Nothing  positive  has,  however,  been 
achieved.  The  first-named  observers  claim  to  have  im- 
munized guinea-pigs  in  whose  blood  an  antitoxin  formed  ; 
the  last-named  thinks  the  serum  of  immunized  horses 
a  specific  for  tuberculosis.  The  field  of  experimentation 
is  an  inviting  one,  though  the  chronic  course  of  the  dis- 
ease lessens  the  certainty  with  which  the  results  can  be 
estimated. 

The  Bacillus  of  Fowl-tuberculosis  (Tuberculosis  gal- 
linarum}. — The  cases  of  tuberculosis  which  occasionally 
occur  spontaneously  in  chickens,  parrots,  ducks,  and 
other  birds  were  originally  attributed  to  the  Bacillus 
tuberculosis,  but  the  recent  works  of  Rivolta,  Mafucci, 
Cadio,  Gilbert,  Roget,  and  others  have  shown  that,  while 
very  similar  in  many  respects  to  the  Bacillus  tuberculosis, 
the  organism  found  in  the  disease  of  birds  has  distinct 
peculiarities  which  make  it  a  different  variety,  if  not  a 


TUBERCULOSIS.  191 

separate  species.  Morphologically,  trie  organisms  are 
.similar,  the  bacillus  of  fowl-tuberculosis  being  a  little 
longer  and  more  slender  than  its  ally. 

Upon  culture-media  a  distinct  rapidity  of  growth  is 
observable,  and  we  find  that,  instead  of  growing  only 
where  glycerin  is  present,  the  Bacillus  tuberculosis  galli- 
narum  will  grow  upon  blood-serum,  agar-agar,  and  bouil- 
lon as  ordinarily  prepared.  It  will  not  grow  upon  potato. 
The  bacillus  will  grow  at  42-43°  C.  quite  as  well  as  at 
37°  C.,  while  the  growth  of  the  tubercle  bacillus  ceases 
at  42°  C.  Moreover,  the  temperature  of  43°  C.  does  not 
attenuate  its  virulence.  The  thermal  death-point  is  70° 
C.  Upon  culture-media  it  can  retain  its  virulence  for 
two  years. 

The  growth  upon  artificial  culture-media  is  luxuriant, 
and  lacks  the  dry  quality  characteristic  of  ordinary 
tubercle-bacillus  cultures.  As  it  becomes  old  a  culture 
of  fowl-tuberculosis  turns  slightly  yellow. 

Birds  are  the  most  susceptible  animals  for  experimental 
inoculation,  the  embryos  and  young  m6re  so  than  the 
adults  ;  guinea-pigs  are  quite  immune.  Artificial  inocu- 
lation can  only  be  made  in  the  subcutaneous  tissue,  never 
through  the  intestine.  The  chief  seat  of  the  disease  is 
the  liver,  where  cellular  nodes,  lacking  the  central  coag- 
ulation and  the  giant-cells  of  mammalian  tuberculosis, 
and  enormously  rich  in  bacilli,  are  found.  The  disease 
never  begins  in  the  lungs,  and  the  fowls  which  are  dis- 
eased never  show  bacilli  in  the  sputum  or  the  dung. 

Rabbits  are  easily  infected,  an  abscess  forming  at  the 
seat  of  inoculation,  and  later  nodules  forming  in  the 
lung,  so  that  the  distribution  is  quite  different  from  that 
seen  in  birds. 

The  bacillus  stains  like  the  tubercle  bacillus,  but  takes 
the  stain  rather  more  easily.  The  resistance  to  acids  is 
about  the  same. 

Pseudo-tuberculosis. — Eberth,  Chantemesse,  Charrin, 
and  Roger  have  reported  certain  cases  of  so-called  pseudo- 
tuberculosis.  The  disease  occurred  spontaneously  in 


192  PATHOGENIC  BACTERIA. 

guinea-pigs,  and  was  characterized  by  the  formation  of 
cellular  nodules  in  the  liver  and  kidneys  much  resembling 
miliary  tubercles.  Cultures  made  from  them  showed  the 
presence  of  a  small  motile  bacillus  which  could  easily  be 
stained  by  ordinary  methods  (Fig.  55).  When  introduced 


FIG.  57. — Bacillus  pseudo-tuberculosis  from  agar-agar;    x  1000  (Itzerott  and 

Niemann). 

subcutaneously  into  guinea-pigs  the  original  disease  was 
produced. 

Pseudo-tuberculosis  seems  to  be  an  indefinite  affection 
of  which  we  have  very  little  knowledge,  and  which  is 
certainly  in  no  way  connected  with  or  related  to  true 
tuberculosis. 


CHAPTER    II. 
LEPROSY. 

L,EPROSY  is  a  disease  of  great  antiquity,  and  very  early 
received  much  attention  and  study.  In  giving  the  laws 
to  Israel,  Moses  included  a  large  number  of  rules  for  its 
recognition,  the  isolation  of  the  sufferers,  the  determina- 
tion of  recovery,  and  observances  to  be  fulfilled  before 
the  convalescent  could  once  more  mingle  with  his  people. 
The  Bible  is  replete  with  accounts  of  miracles  wrought 
upon  lepers,  and  during  the  times  of  biblical  tradition  it 
must  have  been  an  exceedingly  common  and  malignant 
disease. 

At  the  present  time,  although  we  in  the  Northern 
United  States  hear  very  little  about  it,  leprosy  is  still  a 
widespread  disease.  It  exists  in  much  the  same  form  as 
two  thousand  years  ago  in  Palestine,  Syria,  Egypt,  and 
the  adjacent  countries.  It  is  exceedingly  common  in 
China,  Siam,  and  parts  of  India.  Cape  Colony  has  many 
cases.  In  Europe,  Norway,  Sweden,  and  parts  of  the 
Mediterranean  coast  furnish  a  considerable  number  of 
cases.  Certain  islands,  especially  the  Sandwich  Islands,, 
are  regular  hot-beds  for  its  maintenance.  The  United 
States  is  not  exempt,  the  Gulf  coast  being  chiefly  af- 
fected. 

At  one  time  the  view  was  prevalent  that  the  disease 
was  spread  only  by  contagion,  at  another  that  it  was 
miasmatic.  At  present  the  tendency  is  to  view  it  as 
contagious  to  a  degree  rather  less  than  tuberculosis. 
Sometimes  it  is  hereditary. 

The  cause  of  leprosy  is  now  pretty  certainly  deter- 
mined to  be  the  lepra  bacillus  (Fig.  58),  which  was  dis- 

13  193 


i94 


PATHOGENIC  BACTERIA. 


covered  by  Hansen,  and  subsequently  clearly  described 
by  Neisser. 

The  bacillus  is  almost  the  same  size  as  the  tubercle 
bacillus — perhaps  a  little  shorter — but  lacks  the  curve 
which  is  so  constant  in  the  latter.  It  stains  in  very 
much  the  same  way  as  the  tubercle  bacillus,  but  permits 
of  a  rather  more  rapid  penetration  of  the  stain,  so  that 


FIG.  58. — Bacillus   leprse,  seen   in  a  section   through  a  subcutaneous   node ; 
x  500   (Frankel  and  Pfeiffer). 

the  ordinary  aqueous  solutions  of  the  anilin  dyes  color 
it  quite  readily.  It  stains  well  by  Grain's  method, 
by  which  beautiful  tissue  specimens  can  be  prepared. 
The  peculiar  property  of  retaining  the  color  in  the 
presence  of  the  mineral  acids  which  characterizes  the 
tubercle  bacillus  also  characterizes  the  lepra  bacillus, 
and  the  methods  of  Ehrlich,  Gabbett,  and  Unna  can  be 
used  for  its  detection. 

Like  that  of  the  tubercle  bacillus,  its  protoplasm  often 
presents  open  spaces  or  fractures,  which  have  been  re- 


LEPROSY.  195 

garded  by  some  as  spores,  but  which  are  even  less  likely 
to  be  spores  than  the  similar  appearances  in  the  tubercle 
bacillus. 

The  organism  almost  always  occurs  singly  or  in  irreg- 
ular groups,  filaments  being  unknown.  It  is  not  motile. 

Many  experimenters  have  endeavored  to  make  this 
bacillus,  which  is  so  distinctly  present  in  the  nodes  of 
lepra,  grow  upon  artificially-prepared  substances,  but,  in 
spite  of  modern  methods,  improved  apparatus,  and  re- 
fined media,  all,  with  the  exception  of  Bordoni-Uffredozzi, 
have  met  with  failure.  The  observer  named  was  able 
to  grow  upon  a  blood-serum-glycerin  mixture  a  bacillus 
which  partook  of  the  staining  peculiarities  of  the  bacillus 
as  it  appears  in. the  tissues,  but  differed  very  much  in 
morphology.  After  numerous  generations  this  bacillus 
was  induced  to  grow  upon  ordinary  culture-media.  It 
commonly  presented  a  club-like  form,  which  was  re- 
garded by  Baumgarten  as  an  involution  appearance. 
Frankel  points  out  that  the  bacillus  of  Bordoni  is  pos- 
sessed of  none  of  the  essential  characters  of  the  lepra 
bacillus  except  its  staining,  and  does  not  see  in  the  large, 
thick  organism  which  he  cultivated  anything  to  suggest 
the  lepra  bacillus.  Absolute  confirmation  of  the  specific 
nature  of  the  lepra  bacillus  by  means  of  experiments 
upon  animals  is  wanting.  The  lepra  bacillus  not  only 
refuses  to  allow  itself  to  be  cultivated,  but  also  refuses 
to  be  successfully  transplanted  from  animal  to  animal. 
Only  a  very  few  instances  are  recorded  in  which  actual 
inoculation  has  produced  leprosy  in  either  men  or  ani- 
mals. Arning  was  able  to  secure  permission  to  ex- 
periment upon  a  condemned  criminal  in  the  Sandwich 
Islands.  The  man  was  of  a  family  entirely  free  from 
the  disease.  Arning  introduced  beneath  his  skin  frag- 
ments of  tissue  freshly  excised  from  a  lepra  nodule, 
and  kept  the  man  under  observation.  In  the  course 
of  some  months  typical  lesions  began  to  develop  at 
the  points  of  inoculation  and  spread  gradually,  ending 
in  general  lepra  in  the  course  of  about  five  years. 


196  PATHOGENIC  BACTERIA. 

Melcher  and  Artmann  introduced  fragments  of  lepra 
nodules  into  the  anterior  chambers  of  the  eyes  of  rabbits, 
and  observed  the  death  of  the  animals  after  some  months 
with  typical  lepra  lesions  of  all  the  viscera,  especially 
the  cecum. 

While  the  lepra  bacillus  has  much  in  common  with  the 
tubercle  bacillus,  there  is  not  the  slightest  evidence  of 
any  real  identity.  It  has  already  been  shown  that  lepra 
bacilli  do  not  grow  upon  artificial  media,  and  that  they 
cannot  be  readily  transmitted  by  inoculation.  The  fol- 
lowing description  will  show  that  the  relation  of  the 
bacilli  to  the  lesions  is  entirely  different  from  that  of 
the  tubercle  bacilli  to  the  tubercles. 

Like  the  Bacillus  tuberculosis,  the  Bacillus  leprae  proba- 
bly only  occurs  in  places  frequented  by  persons  suffering 
from  the  disease.  That  individuals  are  infected  by  the 
latter  less  readily  than  by  the  former  bacilli  probably 
depends  upon  the  fact  that  leprous  infection  seems  to 
take  place  most  commonly  by  the  entrance  of  the  organ- 
isms into  the  individual  through  cracks  or  fissures  in 
the  skin,  while  the  tuberculous  infection  occurs  through 
the  more  accessible  respiratory  and  digestive  apparatus. 
Once  established  in  the  body,  the  bacillus  by  its  growth 
produces  chronic  inflammatory  nodes — the  analogues  of 
tubercles. 

The  nodes  of  lepra  consist  of  various  kinds  of  cells 
and  of  fibres.  Unlike  the  tubercles,  the  lepra  nodes  are 
vascular,  and  much  of  the  embryonal  tissue  completes 
its  formative  function  by  the  production  of  fibres.  The 
bacilli  are  not  distributed  through  the  nodes  like  tubercle 
bacilli,  but  are  found  in  groups  enclosed  within  the  proto- 
plasm of  certain  large  cells — the  ' '  lepra  cells. ' '  These 
cells  seem  to  be  overgrown  and  partly  degenerated  lym- 
phoid  cells.  Sometimes  they  are  anuclear,  sometimes 
they  contain  several  nuclei  (giant-cells). 

Lepra  nodules  do  not  degenerate  like  tubercles,  and 
the  formation  of  ulcers,  which  constitutes  a  large  part  of 
the  disease,  seems  largely  due  to  the  action  of  external 


LEPROSY.  197 

agencies  upon  the  feebly  vital  pathological  tissue,  which 
is  unable  to  recover  itself  when  injured. 

In  that  form  known  as  anesthetic  leprosy,  nodules  form 
upon  the  peripheral  nerves,  and  by  connective-tissue 
formation,  as  well  as  the  entrance  of  the  bacilli  into  the 
nerve-sheaths,  cause  irritation,  then  degeneration,  of  the 
nerves.  The  anesthesia  which  follows  these  peripheral 
nervous  lesions  is  one  of  the  conditions  predisposing  to 
the  formation  of  ulcers,  etc.  by  allowing  injuries  to  occur 
without  detection  and  to  progress  without  observation. 
The  ulcerations  and  occasional  loss  of  phalanges  that 
follow  these  lesions  occur,  probably,  in  the  same  manner 
as  in  syringomyelia. 

The  disease  advances,  having  first  manifested  itself 
upon  the  face,  extensor  surfaces,  elbows,  and  knees,  to  the 
lymphatics  and  the  internal  viscera.  Death  ultimately 
occurs  from  exhaustion,  if  not  from  the  frequent  inter- 
current  affections  to  which  the  conditions  predispose. 


CHAPTER  III. 

GLANDERS. 

GLANDERS  is  an  infectious  mycotic  disease  which,  very 
fortunately,  is  almost  confined  to  the  lower  animals.  Only 
occasionally  does  it  secure  a  victim  from  hostlers,  drovers, 
soldiers,  and  bacteriologists,  whose  frequent  association 
with  and  experimentation  upon  animals  bring  them  in 
frequent  contact  with  those  which  are  diseased.  Of  all 
the  infectious  diseases  studied  by  scientists,  none  has 
caused  the  havoc  which  glanders  has  wrought.  Several 
men  of  prominence  have  succumbed  to  accidental  in- 
fection. 

Glanders  was  first  known  to  us  as  a  disease  of  the  horse 
and  ass  characterized  by  the  occurrence  of  discrete,  clean- 
ly-cut ulcers  upon  the  mucous  membrane  of  the  nose. 
These  ulcers  are  formed  by  the  breaking  down  of  nodules 
which  can  be  detected  upon  the  diseased  membranes,  and 
show  no  tendency  to  recover,  but  slowly  spread  and  dis- 
charge a  virulent  pus.  The  edges  of  the  ulcers  are  in- 
durated and  elevated,  the  surfaces  often  smooth.  The 
disease  does  not  progress  to  any  great  extent  before  the 
submaxillary  lymphatic  glands  begin  to  enlarge.  Later 
on  these  glands  form  large  lobulated  masses,  which  may 
soften,  open,  and  become  discharging  ulcers.  The  lungs 
may  also  become  infected  by  inspiration  of  the  infectious 
material,  and  contain  small  foci  not  unlike  tubercles  in 
appearance.  The  animals  ultimately  die  of  exhaustion. 

In  1882,  shortly  after  the  discovery  of  the  tubercle 
bacillus,  LofHer  and  Schiitz  discovered  in  the  discharges 
and  tissues  of  this  disease  the  specific  micro-organism, 
the  glanders  bacillus  (Bacillus  mallei ;  Fig.  59),  which  is 
its  cause. 

198 


GLANDERS. 


199 


The  glanders  bacillus  is  somewhat  shorter  and  dis- 
tinctly thicker  than  the  tubercle  bacillus.  It  has  rounded 
ends,  and  it  generally  occurs  singly,  though  upon  blood- 


.    - 


FIG.  59. — Bacillus  mallei,   from  a  culture  upon  glycerin  agar-agar;    x    1000 
(Frankel  and  Pfeiffer). 

serum,  and  especially  upon  potato,  several  joined  indi- 
viduals may  be  found.  Long  threads  are  never  formed. 

The  bacillus  is  non-motile.  Various  observers  have 
claimed  the  discovery  of  spores,  but  although  in  the 
interior  of  the  bacilli  there  have  been  observed  irregular 
spaces  like  the  similar  spaces  in  the  continuity  of  the 
tubercle  bacillus  not  colored  by  the  stains,  they  have 
not  yet  been  definitely  proven  to  be  spores.  The  ob- 
servation of  lyoffler  that  the  bacilli  can  be  cultivated 
after  being  kept  in  a  dry  state  for  three  months  makes  it 
appear  as  if  some  permanent  form  (spore)  occurs.  No 
flagella  have  been  demonstrated  iipon  the  bacillus. 

Like  the  tubercle  bacillus,  the  glanders  bacillus  does 
not  seem  to  find  conditions  outside  the  animal  body  suit- 
able for  its  existence,  and  probably  does  not  occur  except 
as  a  parasite. 

The  organism  only  grows  between  25°  and  42°  C.,  and 
generally  grows  very  slowly,  so  that  attempts  at  its  isola- 


200  PATHOGENIC  BACTERIA. 

tion  and  cultivation  by  the  usual  plate  method  are  apt  to 
fail,  because  the  numerous  other  organisms  in  the  material 
grow  much  more  rapidly. 

The  best  method  of  isolation  seems  to  be  the  use  of  an 
animal  reagent.  It  has  been  said  that  glanders  princi- 
pally affects  horses  and  asses.  Recent  observations,  how- 
ever, have  shown  the  goat,  cat,  hog  (slightly),  field-mouse, 
wood-mouse,  marmot,  rabbit,  guinea-pig,  and  hedgehog 
all  to  be  susceptible  animals.  Cattle,  house-mice,  white 
mice,  and  rats  are  immune. 

The  guinea-pig,  being  a  highly  susceptible  as  well 
as  a  readily  procurable  animal,  naturally  becomes  the 
reagent  for  the  detection  and  isolation  of  the  bacillus. 
When  a  subcutaneous  inoculation  of  some  glanders  pus 
is  made,  the  disease  can  be  observed  in  guinea-pigs 
by  a  tumefaction  in  from  four  to  five  days.  Somewhat 
later  this  tumefaction  changes  to  a  caseous  nodule,  which 
ruptures  and  leaves  a  chronic  ulcer  with  irregular  mar- 
gins. The  lymph-glands  speedily  become  involved,  and 
in  a  month  to  five  weeks  signs  of  general  infection  are 
present.  The  lymph-glands  suppurate,  the  testicles  un- 
dergo the  same  process,  and  still  later  the  joints  exhibit 
a  suppurative  arthritis  containing  the  bacilli.  The  ani- 
mal finally  dies  of  exhaustion.  In  guinea-pigs  no  nasal 
ulcers  form.  In  field-mice,  which  are  even  more  suscepti- 
ble, the  disease  is  much  more  rapid.  No  local  lesions 
are  visible.  In  two  or  three  days  the  animal  seems  un- 
well, the  breathing  is  hurried,  it  sits  still  with  closed 
eyes,  and  without  any  other  preliminaries  tumbles  over 
on  its  side,  dead. 

From  the  tissues  of  the  inoculated  animals  the  pure 
cultures  are  most  easily  made.  Perhaps  the  best  places 
to  secure  the  culture  are  from  softened  nodes  which  have 
not  ruptured  or  from  the  suppurating  joints.  Strauss 
has,  however,  given  us  a  method  which  is  of  great  use, 
because  of  the  short  time  required.  The  material  sus- 
pected to  contain  the  glanders  bacillus  is  injected  into 
the  peritoneal  cavity  of  a  male  guinea-pig.  In  three  or 


GLANDERS.  2OI 

four  days  the  disease  becomes  established.  The  testicles 
enlarge  a  little  ;  the  skin  over  them  becomes  red  and 
shining.  The  testicles  themselves  begin  to  suppurate, 
•and  often  discharge  through  the  skin.  The  animal  dies 
in  about  two  weeks.  If  such  an  animal  be  killed  and  its 
testicles  examined,  the  tunica  vaginalis  testis  will  be 
found  to  contain  pus,  and  sometimes  to  be  partially  ob- 
literated by  inflammatory  exudation.  The  bacilli  are 
present  in  this  pus,  and  can  be  secured  from  it  in  pure 
cultures. 

The  purulent  discharges  from  the  noses  of  horses  and 
from  other  lesions  of  large  animals  generally  contain  very 
few  bacilli,  so  that  a  method  of  isolation  by  an  animal 
is  very  advantageous  by  greatly  increasing  the  number 
of  bacteria. 

The  bacillus  is  an  aerobic  organism,  and  can  be  grown 
in  bouillon,  upon  agar-agar,  better  upon  glycerin  agar- 
agar,  very  well  upon  blood-serum,  and  quite  character- 
istically upon  potato.  It  grows  in  gelatin,  but  this  is 
not  an  appropriate  medium,  because  the  bacillus  develops 
best  at  temperatures  at  which  the  gelatin  is  liquid. 

Upon  4  per  cent,  glycerin  agar-agar  plates  the  colonies 
appear  upon  the  second  day  as  pale-yellow  or  whitish, 
shining  round  dots.  Under  the  microscope  they  appear 
as  brownish-yellow,  thick  granular  masses  with  sharp 
borders. 

The  culture  upon  agar-agar  and  glycerin  agar-agar 
occurs  as  a  moist,  shining  layer  not  possessed  of  distinct 
peculiarities.  Upon  blood-serum  the  growth  is  rather 
characteristic.  The  colonies  along  the  line  of  inoculation 
first  develop  as  circumscribed,  clear,  transparent  drops, 
which  later  become  confluent  and  form  a  transparent 
layer  unaccompanied  by  liquefaction. 

The  most  charcteristic  growth  is  upon  potato.  It  first 
appears  in  about  forty-eight  hours  as  a  transparent, 
honey-like,  yellowish  layer,  developing  only  at  incuba- 
tion-temperature and  soon  becoming  reddish-brown.  As 
this  brown  color  of  the  colony  develops,  the  potato  for 


202  PATHOGENIC  BACTERIA. 

a  considerable  distance  around  it  becomes  greenish- 
brown.  (See  Frontispiece.)  No  other  known  organism 
produces  the  same  appearance  upon  potato. 

The  organism  loses  its  virulence  if  cultivated  for  many 
generations  upon  artificial  media. 

That  this  bacillus  is  the  cause  of  glanders  there  is  no 
room  to  doubt.  L,6ffler  and  Schiitz  have  succeeded  by 
the  inoculation  of  horses  and  asses  in  producing  the 
well-known  disease. 

The  organisms  when  in  cultures  can  be  stained  with 
the  watery  anilin-dye  solutions,  but  are  difficult  to  stain 
in  tissues.  They  do  not  stain  by  Gram's  method. 

The  chief  difficulty  in  staining  the  bacillus  in  tissues 
is  the  readiness  with  which  it  gives  up  the  stain  in  the 
presence  of  decolorizing  agents.  Loffler  at  first  accom- 
plished the  staining  by  allowing  the  sections  to  lie  for 
some  time  (five  minutes)  in  the  alkaline  methylene-blue 
solution,  then  transferring  them  to  a  solution  of  sulphuric 
and  oxalic  acids — 

Concentrated  sulphuric  acid,  2  drops  ; 

5  per  cent,  oxalic-acid  solution,  i  drop  ; 

Distilled  water,  10  c.cm. 

for  five  seconds,  then  transferring  to  absolute  alcohol, 
xylol,  etc.  The  bacilli  appear  dark  blue  upon  a  paler 
ground.  This  method  gives  very  good  results,  but  has 
been  largely  superseded  by  the  use  of  Kuhne's  carbol- 
methylene  blue : 

Methylene  blue,  1.5 

Alcohol,  10. 

5  per  cent,  aqueous  phenol  solution,         100. 

Kiihne's  method  of  staining  is  to  place  the  section  in  the 
stain  for  about  half  an  hour,  wash  in  water,  decolorize 
carefully  in  hydrochloric  acid  (10  drops  to  500  c.cm.  of 
water),  immerse  at  once  in  a  solution  of  lithium  carbonate 
(8  drops  of  a  saturated  solution  of  lithium  carbonate  in  10 
c.cm. of  water),  place  in  a  bath  of  distilled  water  for  a  few 


GLANDERS.  203 

minutes,  dip  into  absolute  alcohol  colored  with  a  little 
methylene  blue,  dehydrate  in  anilin  oil  containing  a 
little  methylene  blue  in  solution,  wash  in  pure  anilin 
oil,  not  colored,  then  in  a  light  ethereal  oil,  clear  in 
xylol,  and  mount  in  balsam. 

When  stained  in  sections  of  tissue  the  bacilli  are 
found  to  occupy  the  interior  of  small  inflammatory  zones 
not  unlike  tubercles  in  appearance.  These  nodules  can 
be  seen  with  the  naked  eye  scattered  through  the  livers, 
kidneys,  and  spleens  of  animals  dead  of  experimental 
glanders.  The  nodules  consist  principally  of  leucocytes, 
but  also  contain  numerous  epithelioid  cells.  As  is  the  case 
with  tubercles,  the  centres  of  the  nodules  are  prone  to 
degenerate,  soften,  and  also  to  suppurate.  The  retro- 
gressive processes  upon  exposed  surfaces,  where  the  break- 
ing down  of  the  nodules  allows  their  contents  to  escape, 
are  the  sources  of  the  typical  ulcerations.  At  times  the 
process  is  progressive,  and  some  of  the  lesions  heal  by 
the  formation  of  a  stellate  scar. 

As  has  been  mentioned,  cultures  of  the  bacillus  lose 
their  virulence  more  or  less  after  four  or  five  generations 
in  artificial  media.  While  this  is  true,  attempts  to  atten- 
uate fresh  cultures  by  heat,  etc.  have  so  far  failed. 

Leo  has  pointed  out  that  white  rats,  which  are  immune 
to  the  disease,  may  be  made  susceptible  by  feeding  with 
phloridzin  and  causing  a  glycosuria. 

Kalning,  Preusse,  Pearson,  and  others  have  pre- 
pared a  substance,  "mallein,"  from  cultures  of  the 
bacillus,  and  suggested  its  employment  for  diagnostic 
purposes.  It  seems  to  be  quite  useful  in  veterinary 
medicine,  the  reaction  occasioned  by  its  injection  being 
similar  to  that  caused  by  the  injection  of  tuberculin  in 
tuberculous  patients.  The  manufacture  of  mallein  is 
not  attended  with  great  difficulty.  The  bacilli  are  grown 
in  glycerin  bouillon  for  several  weeks,  killed  by  heat,  the 
culture  filtered  through  porcelain  and  evaporated  to  one- 
tenth  of  its  volume.  It  has  also  been  prepared  from 
potato  cultures,  which  are  said  to  produce  a  stronger 


204  PA  THOGENIC  BACTERIA . 

toxin.  A  febrile  reaction  of  more  than  1.5°  C.  following 
the  injection  is  said  to  be  specific  of  the  disease.  Babes 
has  asserted  that  the  injection  of  this  toxic  product  into 
susceptible  animals  will  protect  them  from  the  disease. 
Various  experiments  have  been  made  with  curative 
objects  in  view.  Certain  observers  claim  to  have  seen 
good  results  follow  the  injection  of  mallein  in  repeated 
small  doses.  Others,  as  Chenot  and  Picq,  find  the  blood- 
serum  from  immune  animals  like  the  ox  to  be  curative 
when  injected  into  infected  guinea-pigs. 


CHAPTER    IV. 
SYPHILIS. 

ALTHOUGH  syphilis  is  almost  as  well  known  as  it  is 
widespread,  we  have  not  yet  discovered  for  it  a  definite 
specific  cause.  Whether  it  is  due  to  a  protozoan  par- 
asite, or  whether  it  is  due  to  a  bacterium,  the  future 
must  decide.  Numerous  claims  have  been  made  by  those 
whose  studies  have  revealed  organisms  of  one  kind  or 
another  in  syphilitic  tissues,  but  no  one  has  yet  suc- 
ceeded either  in  isolating,  cultivating,  or  successfully  in- 
oculating them. 

In  1884  and  1885,  I^ustgarten  published  a  method  for 
the  staining  of  bacilli  which  he  had  found  in  syphilitic 
tissues  and  assumed  to  be  the  cause  of  the  disease.  The 
staining,  which  is  very  complicated,  requires  that  the 
sections  of  tissue  be  stained  in  Bhrlich's  anilin- water 
gentian-violet  solution  for  twelve  to  twenty-four  hours  at 
the  temperature  of  the -room,  or  for  two  hours  at  40°  C. ; 
washed  for  a  few  minutes  in  absolute  alcohol ;  then  im- 
mersed for  about  ten  seconds  in  a  i  J^  per  cent,  perman- 
ganate-of-potassium  solution,  after  which  they  are  placed 
in  an  aqueous  solution  of  sulphurous  acid  for  one  to  two 
seconds,  thoroughly  washed  in  water,  run  through  alco- 
hol and  oil  of  cloves,  and  finally  mounted  in  Canada 
balsam  dissolved  in  xylol. 

If  the  bacilli  are  supposed  to  be  present  in  pus  or  dis- 
charges from  syphilitic  lesions,  the  cover-glasses  spread 
with  the  material  are  stained  in  the  same  manner,  except 
that  for  the  first  washing  distilled  water  instead  of  abso- 
lute alcohol  is  used. 

This  method  undergoes  a  modification  in  the  hands  of 
De  Giacomi,  who  prefers  to  stain  the  cover-glasses  in  hot 

205 


206  PATHOGENIC  BACTERIA. 

anilin-water-fuchsin  solution  for  a  few  moments,  sections 
in  the  same  solution  cold  for  twenty-four  hours ;  then 
immerse  them  first  in  a  weak,  then  in  a  strong,  solution 
of  chlorid  of  iron.  The  cover-glasses  are  washed  in 
water,  sections  in  alcohol,  and  subsequently  passed 
through  the  usual  reagents  for  dehydration  and  clearing. 


FIG.  60. — Bacillus  of  syphilis  (Lustgarten),  from  a  condyloma;  x  1000  (Itzerott 

and  Niemann). 

In  some  syphilitic  tissues  these  methods  suffice  to  de- 
fine distinct  bacilli  with  a  remarkable  similarity  to  the 
tubercle  bacillus.  The  organism  is  about  the  same  size 
as,  and  even  more  frequently  curved  than,  the  tubercle 
bacillus,  but  often  presents  a  club-like  enlargement  of 
one  end  (involution-form?).  The  bacilli  very  frequently 
occur  singly,  though  more  often  in  groups,  and  never  lie 
free,  but  are  always  enclosed  in  cells.  These  bacilli  are 
not  always  found  in  syphilitic  lesions,  nor  is  their  dem- 
onstration easy  under  the  most  favorable  circumstances. 
Lustgarten  emphasizes  particularly  that  they  are  only 
demonstrable  after  the  most  painstaking  technical  pro- 
cedures. 

The  probability  of  the  specificity  of  this  organism  was 
considerably  lessened  by  the  observation  by  Matterstock, 
Travel,  and  Alvarez  that  in  preputial  smegma,  and  also 


SYPHILIS.  207 

in  vulvar  smegma  from  healthy  individuals,  a  similar 
organism,  identical  both  in  morphology  and  staining 
peculiarities,  could  be  demonstrated.  Of  course  the  oc- 
currence of  Lustgarten's  bacillus  in  the  internal  organs 
could  not  but  argue  against  the  probability  of  its  identity 
with  the  smegma  bacillus  ;  but  L,ustgarten  himself  pointed 
out  that  the  bacilli  of  both  tuberculosis  and  leprosy  stain 
by  his  method,  and  thus  gave  Baumgarten  the  right  to 
suggest  that  the  few  cases  well  adapted  for  the  demon- 
stration of  the  lyustgarten  bacilli  might  be  cases  of  mixed 
infection  of  tuberculosis  and  syphilis. 

The  bacillus  has  not  been  isolated  or  cultivated,  and 
its  proper  relation  to  syphilis  is  a  matter  which  must  be 
decided  by  future  experimentation. 


CHAPTER    V. 
ACTINOMYCOSIS. 

IN  1845,  Langenbeck  discovered  that  the  specific  dis- 
ease of  cattle  known  as  actinomycosis  could  be  com- 
municated to  man.  His  observations,  however,  were  not 
given  to  the  world  until  1878,  one  year  after  Bollinger 
had  discovered  the  cause  of  the  disease  in  animals. 


FIG.  61. — Actinomyces  bovis,  from  the  tongue  of  a  calf;    x  500  (Frankel  and 

Pfeiffer). 

Actinomycosis  is  a  disease  almost  peculiar  to  the  bovine 
animals,  though  sometimes  occurring  in  hogs,  horses, 
men,  and  other  animals. 

The  first  manifestations  of  the  disease  are  usually  found 
either  about  the  jaw  or  in  the  tongue,  in  either  of  which 

208 


ACTINOMYCOSIS.  209 

localities  there  are  produced  considerable  enlargements 
which  are  sometimes  dense  and  fibrous  (wooden  tongue) 
and  sometimes  suppurative.  In  sections  of  these  nodular 
formations  small  yellowish  granules  surrounded  by  some 
pus  can  be  found.  These  granules  when  viewed  beneath 
the  microscope  exhibit  a  peculiar  rosette-like  body — the 
ray-fungus  or  actinomyces. 

The  fungus  is  of  sufficient  size  to  be  detected  by  the 
naked  eye.  It  can  be  colored,  in  sections  of  tissue,  by 
the  use  of  Gram's  method,  or  better  by  Weigert's  fibrin 
stain.  Tissues  pre-stained  with  carmin,  then  stained  by 
Weigert's  method,  give  beautiful  pictures. 

The  entire  fungus-mass  consists  of  several  distinct 
zones  embracing  entirely  different  elements.  At  the 
centre  of  the  mass  there  is  found  a  granular  substance 
containing  numerous  bodies  resembling  micrococci.  Ex- 
tending from  this  centre  into  the  neighboring  tissue  is  a 
radiating,  apparently  branched,  thickly-tangled  mass  of 
mycelial  threads.  These  threads  seem  to  terminate  in 
a  zone  of  conspicuous  club-shaped  radiating  forms  which 
give  the  colonies  the  rosette-like  appearance.  The  cells 
of  the  tissues  affected  and  a  larger  or  smaller  collection 
of  leucocytes  form  the  surrounding  resisting  tissue-zone. 

The  degree  of  chemotactic  influence  exerted  by  the 
organism  seems  to  depend  partly  upon  the  tissue  affected 
and  partly  upon  the  individuality  of  the  animal.  When 
the  animal  is  but  slightly  susceptible,  and  when  the 
tongue  is  the  part  affected,  the  disease  is  characterized 
by  the  production  of  enlargement  due  to  the  formation 
of  cicatricial  tissue.  If,  on  the  other  hand,  the  animal 
is  highly  susceptible  or  the  jaw  is  affected,  the  chief 
symptom  is  suppuration,  with  the  formation  of  cavities 
communicating  by  sinuses. 

Before  the  nature  of  the  affection  was  understood  it 
was  confounded  with  various  diseases  of  the  bones,  prin- 
cipally with  osteosarcoma. 

From  the  tissues  primarily  affected  the  disease  spreads 
to  the  lymphatic  glands,  and  not  infrequently  to  the 

14 


210  PATHOGENIC  BACTERIA. 

lungs.  Israel  has  pointed  out  certain  cases  of  human 
actinomycosis  beginning  in  the  peribronchial  tissues, 
probably  from  inhalation  of  the  fungi. 

The  occurrence  of  three  distinct  elements  as  compo- 
nents of  the  rays  served  to  class  this  organism  among 
the  pleomorphous  bacteria  in  the  genus  Cladothrix, 
where  it  has  remained  undisturbed  for  at  least  a  decade. 
Recent  researches  have,  however,  changed  the  view  held 
by  some  bacteriologists  in  regard  to  the  actinomyces,  and 
caused  them  to  regard  the  organism  as  a  bacillus.  If  it 
be  a  bacillus,  the  central  zone  of  granular  cocci-like 
elements  is  to  be  regarded  as  consisting  of  individuals 
in  process  of  rapid  division  and  spore(?)-formation,  the 
mycelial  zone  as  consisting  of  perfect  individuals,  and 
the  peripheral  zone,  with  the  rosette-like,  club-shaped 
elements,  as  consisting  of  individuals  partly  degener- 
ated through  the  activity  of  the  cells  and  tissue-juices 
(involution-forms). 

Jones  is  of  the  opinion  that  the  disease,  if  not  inden- 
tical  with,  is  closely  allied  to,  tuberculosis,  and  that  the 
occasional  branched  forms  of  tubercle  bacilli  prove  the 
tendency  of  the  individual  bacillus  to  form  a  reticulum. 

When  the  mycelial  threads  are  carefully  examined,  the 
branchings,  which  appear  distinct  upon  hasty  inspection, 
are  found  to  be  more  the  effect  of  a  peculiar  relation 
which  the  threads  bear  to  one  another  than  actual  bifur- 
cations, so  that  it  must  be  regarded  as  very  questionable 
whether  these  threads  ever  so  divide. 

The  organism  may  be  grown  upon  artificial  culture- 
media,  as  has  been  proven  by  Israel  and  Wolff. 

Upon  agar-agar  or  glycerin  agar-agar  it  forms  trans- 
lucent colonies,  about  the  size  of  a  pin's  head,  of  firm, 
almost  cartilaginous,  consistence.  These  colonies  consist 
of  bacillary  individuals,  sometimes  seemingly  branched. 
In  bouillon  similar  dense  globular  organisms  can  be 
grown.  The  blood-serum  colonies,  which  grow  simi- 
larly to  the  agar-agar  colonies,  are  rather  more  luxuri- 
ant, and  slowly  liquefy  the  medium. 


ACTINOMYCOSIS.  211 

When  the  actinomyces  are  grown  upon  artificial  media 
their  virulence  is  retained  for  a  considerable  length  of 
time.  If  introduced  into  the  abdominal  cavities  of  rab- 
bits, there  are  produced  in  the  peritoneum,  mesentery, 
and  omentum  typical  nodules  containing  the  actinomyces 
rays. 

The  organism  can  also  be  grown  in  raw  eggs,  into 
which  it  is  carefully  introduced  through  a  small  opening 
made  under  aseptic  precautions.  In  the  egg  the  organism 
forms  peculiar  long  mycelial  threads  quite  unlike  the  short 
forms  developing  upon  agar-agar. 

The  characteristic  rosettes  which  are  constantly  found 
in  the  tissues  are  never  seen  in  artificial  cultures. 

The  exact  manner  by  which  the  organism  enters  the 
body  is  unknown.  In  some  cases  it  may  be  by  direct 
inoculation  with  pus,  but  there  is  reason  to  believe  that 
the  organism  occurs  in  nature  as  a  saprophyte,  or  as 
an  epiphyte  upon  the  hulls  of  certain  grains,  especially 
barley.  Woodhead  records  a  case  where  a  primary  me- 
diastinal  actinomycosis  in  the  human  subject  was  sup- 
posed to  be  traced  to  perforation  of  the  posterior  pharyn- 
geal  wall  by  a  barley  spikelet  swallowed  by  the  patient. 


CHAPTER  VI. 

MYCETOMA,  OR  MADURA-FOOT. 

A  CURIOUS  disease  of  not  infrequent  occurrence  in  the 
Indian  province  of  Scinde  is  one  known  as  mycetoma, 
Madura-foot,  or  pied  de  Madura.  It  almost  invariably 
affects  natives  of  the  agriculturist  class,  and  in  most 
cases  begins  in  or  is  referred  by  the  patient  to  the  prick 
of  a  thorn.  It  generally  affects  the  foot,  more  rarely 
the  hand,  and  in  one  instance  was  seen  by  Boyce  in  the 
shoulder  and  hip.  It  is  more  common  in  men  than  in 
women,  individuals  between  twenty  and  forty  years  of 
age  suffering  most  frequently,  but  persons  of  any  age  or 
sex  may  suffer  from  the  disease.  It  is  insidious  in  its 
onset,  as  has  been  said,  generally  following  a  slight 
injury,  such  as  the  prick  of  a  thorn.  No  symptoms  are 
observed  in  what  might  be  called  an  incubation  stage  of 
a  couple  of  weeks'  duration,  but  after  this  time  elapses  a 
nodular  growth  gradually  forms,  attaining  in  the  course 
of  time  the  size  of  a  marble.  Its  deep  attachments  are 
indistinct  and  diffuse.  The  skin  becomes  purplish, 
thickened,  indurated,  and  adherent.  The  points  most 
frequently  invaded  at  the  onset  are  the  ball  of  the  great 
toe  and  the  pads  under  the  bases  of  the  fingers  and  toes. 

In  the  course  of  months,  although  progressing  slowly, 
the  lesions  attain  very  perceptible  size,  distinct  tumors 
being  present.  Later,  sometimes  not  until  after  a  year 
or  two,  the  nodes  begin  to  soften,  break  down,  discharge 
their  purulent  contents,  and  originate  ulcers  and  com- 
municating sinuses.  The  discharge  at  this  stage  is  a 
thin  sero-pus,  and  is  always  mixed  with  a  number  of 
fine  round  black  or  pink  bodies,  described,  when  black, 
as  resembling  gunpowder  ;  when  pink,  as  resembling 
212 


MYCETOMA,    OR  MADURA-FOOT.  213 

fish-roe.  It  is  the  detection  of  these  particles  upon 
which  the  diagnosis  rests,  and  upon  which  the  divis- 
ion of  the  disease  into  the  melanoid  and  pale  varieties 
depends. 

The  progress  of  the  disease  causes  an  enormous  size 
and  a  peculiar  deformity  of  the  affected  foot  or  hand. 
The  malady  is  generally  painless. 

The  micro-organismal  nature  of  the  disease  was  early 
suspected.  In  spite  of  the  confusion  caused  by  some 
who  confounded  the  disease  with  and  described  it  as 
''Guinea-worm,"  Carter  held  that  it  was  due  to  some 
indigenous  fungus  as  early  as  1874.  Boyce  and  Surveyor 
believe  that  the  black  particles  of  the  melanoid  variety 
represent  a  curious  metamorphosis  of  a  large  branching 
septate  fungus,  and  that  the  white  particles  of  the  other 
variety  are  the  remains  of  a  lowly-organized  fungus  and 
of  caseous  particles. 

Kanthack  tried  to  prove  the  identity  of  the  fungus 
with  the  well-known  actinomyces,  but  there  seems  to 
be  considerable  doubt  about  the  correctness  of  his  view. 

Vincent  succeeded  in  isolating  the  micro-organism 
by  puncturing  one  of  the  nodes  with  a  sterile  pipette, 
and  has  cultivated  it  upon  artificial  media.  Acid  vege- 
table infusions  seem  suitable  to  its  growth.  It  develops 
scantily  in  bouillon  at  the  room-temperature,  better  at 
37°  C. — in  from  four  to  five  days.  In  twenty  to  thirty 
days  the  colony  attains  the  size  of  a  little  pea. 

In  the  liquid  media  the  colonies  which  cling  to  the 
glass,  and  thus  remain  near  the  surface  of  the  medium, 
develop  a  rose-  or  bright-red  color. 

Cultures  in  gelatin  are  not  very  abundant,  are  colorless, 
and  are  unaccompanied  by  liquefaction. 

Upon  the  surface  of  agar-agar  strikingly  beautiful 
rounded,  glazed  colonies  are  formed.  They  are  at  first 
colorless,  but  later  become  rose-colored  or  bright  red.  The 
majority  of  the  clusters  remain  isolated,  some  of  them 
attaining  the  size  of  a  small  pea.  They  are  generally 
umbilicated  like  a  variola  pustule,  and  present  a  curious 


214 


PATHOGENIC  BACTERIA. 


appearance  when  the  central  part  is  pale  and  the  periphery 
red.  As  the  colony  ages  the  red  color  is  lost  and  it  be- 
comes dull  white.  The  colonies  are  very  adherent  to  the 
surface  of  the  medium,  and  are  said  to  be  of  cartilaginous 
consistence.  The  organism  also  grows  in  milk  without 
coagulation. 

Upon  potato  the  development  is  meagre,  slow,  and 
with  very  little  tendency  to  chromogenesis.  The  color- 
production  is  more  marked  if  the  potato  be  acid  in  reac- 


FIG.  62. — Streptothrix  Madurae  in  a  section  of  diseased  tissue  (Vincent). 

tion.  Some  of  the  colonies  upon  agar-agar  and  potato 
have  a  powdery  surface,  no  doubt  from  the  occurrence  of 
spores.  It  is,  of  course,  an  aerobic  organism. 

Under  the  microscope  the  organism  is  found  by  Vin- 
cent to  be  a  streptothrix — a  true  branched  fungus  con- 
sisting of  long  bacillary  branching  threads  in  a  tangled 
mass.  In  many  of  the  threads  spores  could  be  made  out. 


MYCETOMA,    OR  MADURA-FOOT.  215 

Vincent  was  nnable  to  communicate  the  disease  to  animals 
by  inoculation. 

Microscopic  study  of  the  diseased  tissues  in  cases  of 
mycetoma  is  not  without  interest.  The  healthy  tissue 
is  said  to  be  sharply  separated  from  the  diseased  masses. 
The  latter  appear  as  large  degenerated  tubercles,  except 
that  they  are  extremely  vascular.  The  mycelial  or 
filamentous  fungous  mass  occupies  the  centre  of  the 
degeneration,  where  its  long  filaments  can  be  beautifully 
demonstrated  by  the  use  of  appropriate  stains,  Gram's 
method  being  excellent  for  the  purpose.  The  tissue  sur- 
rounding the  disease-nodes  is  infiltrated  with  small  round 
cells.  The  youngest  nodules  are  seen  to  consist  of  granu- 
lation-tissue, which  in  its  organization  is  checked  by  the 
coagulation-necrosis  which  is  sure  to  overtake  it.  Giant- 
cells  are  few. 

Not  infrequently  small  hemorrhages  occur  from  the 
ulcers  and  sinuses  of  the  diseased  tissues  ;  the  hemor- 
rhages can  be  explained  from  the  abundance  of  small 
blood-vessels  in  the  diseased  tissue. 

Although  the  disease  has  been  described  as  occurring 
in  Scinde,  it  is  not  limited  to  that  province,  having  been 
met  with  in  Madura,  Hissar,  Bicanir,  Dehli,  Bombay, 
Baratpur,  Morocco,  Algeria,  one  case  by  Bastini  and 
Campana  in  Italy,  and  one  by  Kempner  in  America. 


CHAPTER    VII. 

FARCIN  DU  BCEUF. 

THE  peculiar  disease  which  sometimes  affects  numbers 
of  cattle  in  Guadeloupe,  and  which  was  described  by 
the  older  writers  as  farcin  du  bceuf,  has  been  carefully 
studied  by  Nocard.  It  is  a  disease  of  cattle  character- 
ized by  a  superficial  lymphangitis  and  lymphadenitis, 
affecting  the  tracheal,  axillary,  prescapular,  and  other 
glands.  The  affected  glands  enlarge,  suppurate,  and 
discharge  a  creamy,  sometimes  a  grumous,  pus.  The 
internal  organs  are  often  affected  with  a  pseudo-tubercu- 
losis whose  central  areas  undergo  a  purulent  or  caseous 
degeneration. 

In  the  researches  of  Nocard  it  was  discovered,  by 
staining  by  Gram's  and  by  Kiihne's  methods,  that  in 
the  centres  of  the  tubercles  micro-organisms  could  be 
defined.  They  resembled  long  delicate  filaments  rather 
intricately  woven,  characterized  by  distinct  ramifications 
which  made  clear  the  proper  classification  of  the  organ- 
ism as  a  streptothrix.  The  organism  was  successfully 
cultivated  by  Nocard  upon  various  culture-media  at  the 
temperature  of  the  body.  It  is  aerobic. 

In  bouillon  the  organism  develops  in  the  form  of  color- 
less masses  irregular  in  size  and  shape,  some  of  which 
float  upon  the  surface,  others  of  which  sink  to  the  bottom 
of  the  liquid.  Sometimes  the  surface  is  covered  by  an 
irregular  fenestrated  pellicle  of  a  gray  color. 

Upon  agar-agar  the  growth  develops  in  small,  rather 
discrete,  irregularly  rounded,  opaque  masses  of  a  yellow- 
ish-white color.  The  surfaces  of  the  colonies  are  tuber- 
culated,  and  an  appearance  somewhat  like  a  lichen  is 
observed. 

216 


FARCIN  DU  BCEUF.  217 

Upon  potato  very  dry  scales  of  a  pale-yellow  color 
rapidly  develop.  , 

The  growth  upon  blood-serum  is  less  luxuriant,  but 
similar  to  that  upon  agar-agar. 

In  milk  the  organism  produces  no  coagulation  by  its 
growth,  and  does  not  alter  the  reaction. 

Microscopic  study  always  reveals  the  organism  as  the 
same  tangled  mass  of  filaments  seen  in  the  tissues.  The 
old  cultures  are  rich  in  spores,  which  are  very  small  and 
develop  upon  the  most  superficial  portions  of  the  growth. 
These  spores  resist  the  penetration  of  stains  to  a  rather 
unusual  extent. 

Cultures  retain  their  virulence  for  a  long  time  :  Nocard 
found  one  virulent  after  it  had  been  kept  for  four  months 
in  an  incubating  oven  at  40°  C. 

The  streptothrix  of  farcin  du  bceuf  is  pathogenic  for 
guinea-pigs,  cattle,  and  sheep ;  dogs,  rabbits,  horses,  and 
asses  are  immune. 

When  the  culture  or  some  pus  containing  the  micro- 
organism is  injected  subcutaneously  into  a  guinea-pig,  a 
voluminous  abscess  results.  Not  long  afterward  the  lym- 
phatic vessels  and  glands  of  the  region  are  the  seat  of  swell- 
ing and  induration,  and  extensive  phlegmons  form,  which 
rupture  externally  and  discharge  considerable  pus.  The 
animal,  of  course,  becomes  extremely  ill  and  seems  about 
to  die ;  instead,  it  slowly  recovers  its  nonsnal  condition. 

In  other  animals,  as  the  cow  and  the  sheep,  the  subcu- 
taneous inoculation  results  in  an  abscess  relatively  less 
extensive.  This  ulcerates,  then  indurates,  and  seems  to 
disappear,  but  after  the  lapse  of  several  weeks  or  months 
opens  again  in  the  form  of  a  new  abscess. 

In  animals  which  are  immune  or  nearly  immune,  like 
the  horse,  the  ass,  the  dog,  and  the  rabbit,  the  subcuta- 
neous inoculation  is  followed  by  the  formation  of  a  small 
abscess  which  speedily  cicatrizes. 

Intraperitoneal  inoculation  in  the  guinea-pig  gives  rise 
to  an  appearance  resembling  tuberculosis.  The  omen  turn 
may  be  extensively  involved  and  full  of  softened  nodes. 


2i8  PATHOGENIC  BACTERIA. 

The  liver,  spleen,  and  kidneys  appear  full  of  tuberclesy 
but  careful  examination  will  satisfy  the  observer  that 
the  tubercles  are  only  upon  the  peritoneal  surfaces,  not 
in  the  organs. 

Intravenous  introduction  of  the  cultures  produces  a 
condition  much  resembling  general  miliary  tuberculosis. 
All  the  organs  contain  the  pseudo-tubercles  in  consider- 
able numbers. 


CHAPTER  VIII. 
RHINOSCLEROMA.  '• 

IN  Austria,  Hungary,  Italy,  and  some  parts  of  Ger- 
many there  sometimes  occurs  a  peculiar  disease  of  the 
anterior  nares,  characterized  by  the  occurrence  of  circum- 
scribed tumors,  known  as  rhinoscleroma.  The  tumor- 
masses  are  somewhat  flattened,  isolated  or  coalescent, 
grow  with  great  slowness,  and  recur  if  excised.  The  dis- 
ease commences  in  the  mucous  membrane  and  the  adjoin- 
ing skin,  and  spreads  to  the  skin  in  the  neighborhood  by 
a  slow  invasion,  involving  the  upper  lip,  jaw,  hard  palate, 
and  sometimes  the  pharynx.  The  growths  are  without 
evidences  of  inflammation,  do  not  ulcerate,  and  consist 
microscopically  of  infiltration  of  the  papilla  and  corium 
of  the  skin,  with  round  cells  which  change  in  part  to 
fibrillar  tissue.  The  tumors  possess  a  well-developed 
lymph-vascular  system.  Sometimes  the  cells  undergo 
hyaline  degeneration. 

In  these  little  tumors  the  researches  of  Von  Frisch  dis- 
covered little  bacilli  much  resembling  both  in  morphol- 
ogy and  vegetation  the  pneumo-bacilli  of  Friedlander, 
and,  like  them,  surrounded  by  capsules.  The  only 
marked  difference  between  the  so-called  bacillus  of  rhi- 
noscleroma and  the  Bacillus  pneumonise  of  Friedlander 
is  that  the  former  stains  well  by  Gram's  method,  while 
the  latter  does  not,  and  that  the  former  is  rather  more 
distinctly  rod-shaped  than  the  latter,  and  more  often 
shows  its  capsule  in  culture-media. 

The  bacillus  can  easily  be  cultivated,  and  in  all  media 
resembles  the  bacillus  of  Friedlander  too  closely  to  be 
distinguished  from  it.  Even  when  inoculated  into  animals 
the  bacillus  behaves  much  like  Friedlander' s  bacillus. 

Inoculation  has,  so  far,  failed  to  produce  the  disease 
either  in  men  or  in  the  lower  animals. 

219 


B.     THE  TOXIC   DISEASES. 


CHAPTER   I. 
DIPHTHERIA. 

IN  1883,  Klebs  pointed  out  the  existence  of  a  bacillus 
in  the  pseudo-membranes  upon  the  fauces  of  patients 
suffering  from  diphtheria,  but  it  was  not  until  1884  that 
L/offler  succeeded  in  isolating  and  cultivating  the  organ- 
ism, which  is  now  known  by  both  their  names — the 
Klebs-Loffler  bacillus. 

The  bacillus  as  described  by  LofHer  is  about  the  length 
of  the  tubercle  bacillus,  about  twice  its  diameter,  has  a 

** 

**  / 


i*  *%& 


r-  g£  wta 

i  ,c-t  ^ 

*  "1^.  V 


rVf 

fy\^\, 
*~£>      ^ 


FIG.  63. — Bacillus   diphtheria,  from   a   culture   upon   blood-serum;     x  1000 
(Frankel  and  Pfeififer). 

curve  similar  to  that  which  characterizes  the  tubercle 
bacillus,  and  has  rounded  ends  (Fig.  63).     It  does  not 
form  chains,  though  two,  three,  and  rarely  four  individ- 
220 


DIPHTHERIA.  221 

uals  may  be  found  joined ;  generally  the  individuals  are 
all  separate  from  one  another.  The  morphology  of  the 
bacillus  is  peculiar  in  its  considerable  irregularity,  for 
among  the  well-formed  individuals  which  abound  in 
fresh  cultures  a  large  number  of  peculiar  organisms  are 
to  be  found,  some  much  larger  than  normal,  some  with 
one  end  enlarged  to  a  club-shape,  some  greatly  elongated, 
with  both  ends  expanded  into  club-shaped  enlargements. 
These  bizarre  forms  seem  to  represent  an  involution-form 
of  the  organism,  for,  while  present  in  perfectly  fresh  cul- 
tures, they  are  so  abundant  in  old  cultures  that  scarcely 
a  single  well-formed  bacillus  can  be  found.  It  not  infre- 
quently happens  that  in  unstained  bacilli  distinct  gran- 
ules can  be  defined  at  the  ends — polar  granules — thus 
giving  the  organism  somewhat  the  appearance  of  a 
diplococcus. 

The  bacillus  can  be  readily  stained  by  aqueous  solu- 
tions of  the  anilin  colors,  but  more  beautifully  and 
characteristically  with  L,6fHer's  alkaline  methylene  blue  : 

Saturated  alcoholic  solution  of  methylene  blue,    30  ; 
i :  10,000  aqueous  solution  of  caustic  potash,     100  ; 

and  an  aqueous  solution  of  dahlia,  as  recommended  by 
Roux. 

When  cover-glass  preparations  are  stained  with  these 
solutions,  the  bizarre  forms  already  mentioned  are  much 
more  obvious  than  in  the  unstained  individuals,  and 
the  contrast  between  the  polar  granules,  which  color  in- 
tensely, and  the  remainder  of  the  bacillus,  which  tinges 
slightly,  is  marked.  Through  good  lenses  the  organisms 
are  always  distinct  bacilli,  notwithstanding  the  fact  that 
the  ends  stain  more  deeply  than  the  centres,  and  it  is 
only  through  poor  lenses  that  the  organisms  can  be  mis- 
taken for  diplococci.  The  bacilli  stain  well  by  Gram's 
method,  this  being  a  good  method  to  employ  for  their 
definition  in  sections  of  tissue,  though  Welch  and  Abbott 
assert  that  Weigert's  fibrin  method  and  picro-carmin  give 
the  most  beautiful  results. 


222  PATHOGENIC  BACTERIA. 

The  diphtheria  bacillus  does  not  form  spores,  and  is 
delicate  in  its  thermal  range.  L,6ffler  found  that  it  could 
not  endure  a  temperature  of  60°  C.,  and  Abbott  has  shown 
that  a  temperature  of  58°  C.  for  ten  minutes  is  fatal  to  it. 
Notwithstanding  this  susceptibility,  the  organism  can 
be  kept  alive  for  several  weeks  after  being  dried  upon 
shreds  of  silk  or  when  surrounded  by  dried  diphtheria 
membrane. 

No  flagella  have  been  demonstrated  upon  the  bacillus. 
It  is  non-motile. 

Fernbach  has  shown  that  when  the  organisms  are 
grown  in  a  medium  exposed  to  a  passing  current  of  air, 
the  luxuriance  of  their  development  is  increased,  though 
their  life-cycle  is  shorter.  The  growth  can  also  take 
place  when  the  air  is  excluded,  so  that  the  bacillus  must 
be  classed  among  the  optional  anaerobic  organisms. 

The  diphtheria  bacillus  grows  readily  upon  all  the 
ordinary  media,  and  is  a  very  easy  organism  to  obtain 
in  pure  culture.  Loffler  has  shown  that  the  addition 
of  a  small  amount  of  glucose  to  the  culture-medium 
increases  the  rapidity  of  the  growth,  and  suggests  a 
special  medium  which  bears  his  name — LofHer's  blood- 
serum  mixture : 

Blood-serum,  3 ; 

Ordinary  bouillon  +  i  per  cent,  of  glucose,     i. 

This  mixture  is  filled  into  tubes,  coagulated,  and  steril- 
ized like  blood-serum,  and  is  one  of  the  best-known  media 
in  connection  with  the  study  of  diphtheria. 

The  clinical  impossibility  of  making  an  accurate  diag- 
nosis of  diphtheria  without  a  bacteriologic  examination 
has  made  many  private  physicians  and  many  medical 
societies  and  boards  of  health  equip  laboratories  where 
accurate  examinations  can  be  made.  The  method  re- 
quires some  apparatus,  though  a  competent  bacteriologist 
can  often  make  shift  with  a  bake-oven,  a  wash-boiler, 
and  other  household  furniture  instead  of  the  regular 
sterilizers  and  incubators,  which  are  expensive. 


DIPHTHERIA.  223 

When  it  is  desired  to  make  a  bacteriologic  diagnosis 
of  a  suspected  case  of  diphtheria  or  to  secure  the  bacillus 
in  pure  culture,  a  sterile  platinum  wire  having  a  small 
loop  at  the  end,  or  a  swab  made  by  wrapping  a  little 
•cotton  around  •  the  end  of  a  piece  of  wire  and  carefully 
sterilizing  in  a  test-tube,  is  introduced  into  the  throat 
and  touched  to  the  false  membrane,  after  which  it  is 
smeared  carefully  over  the  surface  of  at  least  three  of 
the  blood-serum-mixture  tubes,  without  either  again 
touching  the  throat  or  being  sterilized.  The  tubes  thus 
inoculated  are  stood  away  in  an  incubating  oven  at  the 
temperature  of  37°  C.  for  twelve  hours,  then  examined. 
If  the  diphtheria  bacillus  is  present  upon  the  first  and 
second  tubes,  there  will  be  a  smeary  yellowish-white  layer, 
with  outlying  colonies  on  the  second  tube,  while  the  third 
tube  will  show  rather  large  isolated  whitish  or  slightly 
yellowish  colonies,  smooth  in  appearance,  but  rather  ir- 
regular in  outline.  Very  often  the  colonies  are  china- 
white  in  appearance.  These  colonies,  if  found  by  micro- 
scopic examination  to  be  made  up  of  diphtheria  bacilli, 
will  confirm  the  diagnosis  of  diphtheria,  and  will  at  the 
same  time  give  pure  cultures  when  transplanted.  There 
are  very  few  other  bacilli  which  grow  so  rapidly  upon 
LofHer's  mixture,  and  scarcely  one  other  which  is  found 
in  the  throat. 

Ohlmacher  recommends  the  microscopic  examination 
of  the  still  invisible  growth  in  five  hours.  A  platinum 
loop  is  rubbed  over  the  inoculated  surface  ;  the  material 
secured  is  then  mixed  with  distilled  water,  dried  on  a 
cover-glass,  stained  with  methylene  blue,  and  examined. 
This  method,  if  reliable,  will  be  very  valuable  in  making 
an  early  diagnosis  preparatory  to  the  use  of  the  antitoxin. 

The  presence  of  diphtheria  bacilli  in  material  taken 
from  the  throat  does  not  necessarily  prove  the  patient  to 
be  diseased.  Virulent  bacilli  can  often  be  discovered  in 
the  throats  of  healthy  persons  who  have  knowingly  or 
unknowingly  come  in  contact  with  the  disease.  The 
bacteriologic  examination  is  only  an  adjunct  to  the 


224  PATHOGENIC  BACTERIA. 

clinical  diagnosis,  and  must  never  be  taken  as  positive 
in  itself. 

The  bacillus  grows  similarly  upon  blood-serum  and 
Loffler's  mixture.  Upon  glycerin  agar-agar  and  agar-agar 
the  colonies  are  much  larger,  more  translucent,  always 


a  b  c 

FIG.  64. — Diphtheria  bacilli  (from  photographs  taken  by  Prof.  E.  K.  Dun- 
ham, Carnegie  Laboratory,  New  York) :  a,  pseudo-bacillus ;  b,  true  bacillus ; 
c,  pseudo-bacillus. 

without  the  yellowish-white  or  china-white  color  of  the 
blood-serum  cultures,  and  generally  are  distinctly  divided 
into  a  small  elevated  centre  and  a  flatter  surrounding  zone 
with  indented  edges,  sometimes  with  a  distinctly  radiated 
appearance.  It  must  be  remarked  that  when  sudden 
transplantations  are  made  from  blood-serum  to  agar- 
agar  the  growth  resulting  is  meagre,  but  the  oftener 
this  growth  is  transplanted  to  fresh  agar-agar  the  more 
luxuriant  it  becomes. 


DIPHTHERIA.  22$ 

The  growth  in  gelatin  puncture-cultures  is  character- 
ized by  small  spherical  colonies  which  develop  along  the 
entire  length  of  the  needle-track.  The  gelatin  is  not 
liquefied. 

Upon  the  surface  of  gelatin  plates  the  colonies  that 
develop  do  not  attain  anything  like  the  size  of  the  colo- 
nies upon  Loffler's  mixture.  They  appear  to  the  naked 


FIG.   65. — Bacillus  diphtherise,  colony  twenty-four  hours  old  upon  agar-agar; 
x  100  (Frankel  and  Pfeiffer). 

eye  as  whitish  points  with  smooth  contents  and  regular 
though  sometimes  indented  borders.  Under  the  micro- 
scope they  appear  as  granular,  yellowish-brown  colonies 
with  irregular  borders  (Fig.  65). 

When  planted  in  bouillon  the  organism  causes  a  diffuse 
cloudiness  at  first,  but,  not  being  motile,  soon  settles  to 
the  bottom  in  the  form  of  a  rather  flocculent  precipitate 
which  has  a  tendency  to  cling  to  the  sides  of  the  glass. 
Sometimes  a  delicate  irregular  mycoderma  forms  upon 
the  surface,  especially  when  the  cultivation  is  made  by 
the  method  of  Fernbach  with  a  passing  current  of  air. 
This  mycoderma,  which  may  appear  quite  regular  when 
the  flask  is  undisturbed,  is  so  brittle  that  it  at  once  falls 
to  pieces  if  the  flask  be  moved. 

15 


226  PATHOGENIC  BACTERIA. 

Spronck  has  recently  determined  that  the  characteris- 
tics of  the  growth  of  the  diphtheria  bacillus  in  bouillon, 
as  well  as  the  amount  of  toxin-production,  vary  accord- 
ing to  the  amount  of  glucose  in  the  bouillon.  He  divides 
the  cultures  into  three  types  : 

Type  A.  The  reaction  of  the  bouillon  becomes  acid 
and  remains  acid,  the  acidity  increasing.  The  bacilli 
accumulate  at  the  bottom  of  the  clear  liquid.  The 
toxin-production  is  meagre. 

Type  B.  There  is  no  change  from  alkalinity  to  acidity, 
but  the  original  alkalinity  of  the  bouillon  steadily  in- 
creases. The  culture  is  very  rich,  the  bottom  of  the 
flask  shows  a  considerable  sediment,  the  liquid  is  cloudy, 
and  a  delicate  growth  occupies  the  surface.  The  toxicity 
is  very  great. 

Type  C.  In  a  few  days  the  reaction  of  the  culture 
becomes  acid,  and  then  later  on  changes  to  alkaline. 
During  the  acid  period  the  liquid  is  clear,  with  a  white 
surface-growth.  When  the  alkalinity  returns  the  bouillon 
clouds  and  the  surface-growth  increases  in  thickness. 
Sediment  accumulates  at  the  bottom  of  the  flask.  The 
toxicity  of  the  culture  is  much  less  than  in  Type  B. 

Spronck  regards  the  varying  reaction  as  due  to  the 
fermentation  of  the  glucose,  and  asserts  that  the  most 
luxuriant  and  toxic  cultures  are  those  in  which  no 
glucose  is  present.  To  exclude  as  much  of  the  undesir- 
able sugar  as  possible,  he  makes  the  bouillon  from  the 
stalest  meat  obtainable,  preferring  it  when  just  about  to 
putrefy.  Of  the  meats  experimented  with,  beef  was 
found  to  be  the  best. 

Upon  potato  the  bacillus  only  develops  when  the  reac- 
tion is  alkaline.  The  potato  growth  is  not  characteristic. 
Welch  and  Abbott  always  secured  a  growth  of  the  organ- 
ism when  planted  upon  potato,  but  do  not  mention  the 
reaction  of  the  medium  they  employed. 

Milk  is  an  excellent  medium  for  the  cultivation  of  the 
Bacillus  diphtheriae,  and  is  possibly  at  times  a  means  of 
infection.  L,itmus  milk  is  an  excellent  medium  for  ob- 


DIPHTHERIA.  227 

serving  the  changes  of  reaction  brought  about  by  the 
growth  of  the  bacillus.  At  first  the  alkalinity,  which 
is  always  favorable  to  the  development  of  the  bacillus, 
is  destroyed  by  the  production  of  an  acid.  When  the 
culture  is  old  the  acid  is  replaced  by  a  strong  alkaline 
reaction. 

Diphtheria  as  it  occurs  in  man  is  generally  a  disease 
characterized  by  the  formation  of  a  pseudo-membrane 
upon  the  fauces.  It  is  a  local  infection,  due  to  the 
presence  and  development  of  the  bacilli  on  the  pseudo- 
membrane,'  but  is  accompanied  by  a  general  toxemia 
resulting  from  the  absorption  of  a  violently  poisonous 
substance  produced  by  the  bacilli.  The  bacilli  are  found 
only  in  the  membranous  exudation,  and  most  plentifully 
in  its  older  portions.  As  a  rule,  they  do  not  distribute 
themselves  through  the  circulation  of  the  animal,  though 
at  times  they  may  be  found  in  the  heart's  blood. 

The  most  malignant  cases  of  the  disease  seem  to  be 
due  to  pure  infection  by  the  diphtheria  bacillus,  though 
such  cases  are  more  rare  than  those  in  which  the  Strepto- 
coccus pyogenes  and  Staphylococcus  aureus  and  albus  are 
found  in  association  with  it. 

It  may  be  well  to  remark  that  all  pseudo-membranous 
diseases  of  the  throat  are  not  diphtheria,  but  that  some 
of  them  result  from  the  activity  of  the  pyogenic  organ- 
isms alone. 

No  more  convincing  proof  of  the  existence  of  a  power- 
ful poison  in  diphtheria  could  be  desired  than  the  evi- 
dences of  general  toxemia  resulting  from  the  absorption 
of  material  from  a  comparatively  small  number  of  bacilli 
situated  upon  a  little  patch  of  mucous  membrane. 

In  animals  artificially  inoculated  the  lesions  produced 
are  not  identical  with  those  seen  in  the  human  subject, 
yet  they  have  the  same  general  features  of  local  infection 
with  general  toxemia. 

Guinea-pigs,  kittens,  and  young  pups  are  susceptible 
animals.  When  half  a  cubic  centimeter  of  a  twenty-four- 
hour-old  bouillon  culture  is  injected  beneath  the  skin  of 


228  PATHOGENIC  BACTERIA. 

such  an  animal,  the  bacilli  multiply  at  the  point  of  in- 
oculation, with  the  production  of  a  patch  of  inflamma- 
tion associated  with  a  distinct  fibrinous  exudation  and 
the  presence  of  an  extensive  edema.  The  animal  dies  in 
twenty-four  to  thirty-six  hours.  The  liver  is  enlarged, 
and  sometimes  shows  minute  whitish  points,  which  in 
microscopic  sections  prove  to  be  necrotic  areas  in  which 
the  cells  are  completely  degenerated  and  the  chromatin  of 
their  nuclei  is  scattered  about  in  granular  form.  Similar 
necrotic  foci,  to  which  attention  was  first  called  by  Oertel, 
are  present  in  nearly  all  the  organs  in  cases  of  death  from 
the  toxin.  The  bacilli  are  constantly  absent  from  these 
lesions.  Flexner  has  shown  these  foci  to  be  common  to 
numerous  irritant  poisonings,  and  not  peculiar  to  diph- 
theria alone. 

The  lymphatic  glands  are  usually  enlarged  ;  the  adrenals 
are  also  enlarged,  and,  in  cases  into  which  the  live  bacilli 
have  been  injected,  are  hemorrhagic. 

Sometimes  the  bacilli  themselves  are  present  in  the 
internal  organs,  and  even  in  the  blood,  but  generally  this 
is  not  the  case. 

It  might  be  argued,  from  the  different  clinical  pictures 
presented  by  the  disease  as  it  occurs  in  man  and  in 
animals,  that  they  were  not  expressions  of  the  same 
thing.  A  careful  study,  however,  together  with  the  evi- 
dences adduced  by  Roux  and  Yersin,  who  found  that 
when  the  bacilli  were  introduced  into  the  trachea  of 
animals  opened  by  operation  a  typical  false  membrane 
was  produced,  and  that  diphtheritic  palsy  often  followed, 
and  of  hundreds  of  investigators,  who  find  the  bacilli 
constantly  present  in  the  disease  as  it  occurs  in  man, 
must  satisfy  us  that  the  doubt  of  the  etiological  role  of 
the  bacillus  rests  on  a  very  slight  foundation. 

One  reason  for  skepticism  in  this  particular  is  the 
supposed  existence  of  a  pseudo-diphtheria  bacillus,  which 
has  so  many  points  in  common  with  the  real  diphtheria 
bacillus  that  it  is  difficult  to  distinguish  between  them. 
We  have,  however,  come  to  regard  this  pseudo-bacillus  as 


DIPHTHERIA.  229 

an  attenuated  form  of  the  real  bacillus.  The  chief  points 
of  difference  between  the  bacilli  are  that  the  pseudo- 
bacillus  is  shorter  than  the  diphtheria  bacillus  when 
grown  upon  blood-serum  ;  that  the  cultures  in  bouillon 
progress  much  more  rapidly  at  a  temperature  of  from 
20-22°  C.  than  those  of  the  true  bacillus ;  and  that  the 
pseudo-bacillus  is  not  pathogenic  for  animals.  These 
slight  distinctions  are  all  exactly  what  might  be  expected 
of  an  organism  whose  virulence  had  been  lost,  and  whose 
vegetative  powers  had  been  altered,  by  persistent  manip- 
ulation or  by  unfavorable  surroundings. 

The  diphtheria  bacilli  are  always  present  in  the  throats 
of  patients  suffering  from  diphtheria,  and  constitute  the 
element  of  contagion  by  being  accidentally  discharged 
by  the  nose  or  mouth  by  coughing,  sneezing,  vomiting, 
etc.  Whoever  comes  in  contact  with  such  material  is  in 
danger  of  infection. 

It  is  of  great  interest  to  notice  the  remarkable  results 
obtained  by  Biggs,  Parke,  and  Beebe  in  New  York,  where 
the  bacteriological  examinations  conducted  in  connection 
with  diphtheria  show  that  the  virulent  bacilli  may  be 
found  in  the  throats  of  convalescents  as  long  as  five  weeks 
after  the  discharge  of  the  membrane  and  the  commence- 
ment of  recovery,  and  that  they  exist  not  only  in  the 
throats  of  the  patients  themselves,  but  also  in  the  throats 
of  their  care-takers,  who,  while  not  themselves  infected, 
may  be  the  means  of  conveying  the  disease  from  the 
sick-room  to  the  outer  world.  The  importance  of  this 
observation  must  be  apparent  to  all  readers,  and  serves 
as  further  evidence  why  most  thorough  isolation  should 
be  practised  in  connection  with  this  dreadful  disease. 

From  time  to  time  reference  has  been  made  to  the 
toxin  elaborated  by  the  diphtheria  bacillus.  Roux  and 
Yersin  first  demonstrated  the  existence  of  this  substance 
in  cultures  passed  through  a  Pasteur  porcelain  filter. 
The  toxin  is  intensely  poisonous,  and  by  the  modern 
improved  methods  can  be  secured  in  such  concentration 
that  o.  i  c.cm.  will  kill  a  guinea-pig  in  twenty-four  hours. 


230  PATHOGENIC  BACTERIA, 

The  toxin  is  not  an  albuminous  substance,  and  can  be 
elaborated  by  the  bacilli  when  grown  in  non-albuminous 
urine,  or,  as  suggested  by  Uschinsky,  in  non-albuminous 
solutions  whose  principal  ingredient  is  asparagin.  The 
toxic  value  of  the  cultures  is  greatest  in  the  second  or 
third  week. 

In  addition  to  the  toxin,  a  toxalbumin  has  been  isolated 
by  Brieger  and  Frankel. 

Behring  discovered  that  the  blood  of  animals  rendered 
immune  to  diphtheria  by  inoculation,  first  with  attenu- 
ated and  then  with  virulent  organisms,  contained  a  neu- 
tralizing substance  which  was  capable  of  annulling  the 
effects  of  the  bacilli  or  the  toxin  when  simultaneously  or 
subsequently  inoculated  into  non-protected  animals.  This 
substance,  in  solution  in  the  blood-serum  of  the  immu- 
nized animals,  is  the  diphtheria  antitoxin. 

The  preparation  of  the  antitoxin  for  therapeutic  pur- 
poses received  a  further  elaboration  in  the  hands  of  Roux. 
The  subject  is  one  of  great  interest,  but  must  be  consid- 
ered briefly  in  a  work  of  this  kind. 

The  antitoxin  is  manufactured  commercially  at  present, 
the  method  being  the  immunization  of  large  animals  to 
great  quantities  of  the  toxin,  and  the  withdrawal  of  their 
antitoxic  blood  when  the  proper  degree  of  immunity  has 
been  attained.  The  details  are  as  follows  : 

The  Preparation  of  the  Toxin. — The  method  employed 
by  Roux  and  others  at  the  present  time  was  first  sug- 
gested by  Fernbach,  and  consists  in  growing  the  most 
virulent  bacilli  obtainable  in  alkaline  bouillon  exposed 
in  a  thin  layer  to  the  passage  of  a  current  of  air. 

The  cultures  are  allowed  to  grow  for  three  or  four 
weeks  at  a  temperature  of  37°  C.,  with  a  stream  of 
moist  air  constantly  passing  over  them.  After  the  given 
time  has  passed,  it  will  be  found  that  the  acidity  prima- 
rily produced  by  the  bacillus  gives  place  to  a  much  more 
intense  alkalinity  than  originally  existed.  The  acme  of 
the  toxin-production  seems  to  keep  pace  with  this  alka- 
line production.  When  u  ripe,"  0.4  per  cent,  of  trikresol 


DIPHTHERIA.  231 

is  added  to  the  cultures,  which  are  then  filtered  through 
porcelain.  If  the  toxin  must  be  kept  before  using,  it  is 
best  to  preserve  it  unfiltered,  as  it  deteriorates  more  rap- 
idly after  nitration.  Unfiltered  toxin  causes  too  much 
local  irritation.  If  the  bacillus  employed  was  virulent 
and  the  conditions  of  culture  were  favorable,  the  filtered 
culture  should  be  so  toxic  that  o.  i  c.cm.  would  be  fatal  to 
a  5oogram  guinea-pig  in  twenty-four  hours  (Roux).  Even 
under  the  most  favorable  circumstances  it  is  difficult  to 
obtain  a  toxin  which  will  kill  in  less  than  thirty  hours. 

The  experience  of  the  author  with  Fernbach's  appara- 
tus has  not  been  satisfactory.  The  passing  current  of  air 
is  a  frequent  source  of  contamination  to  the  culture,  and 
requires  great  care.  In  the  end  it  is  questionable  whether 
the  toxin  thus  produced  is  better  than  that  obtained  from 
an  ordinary  flask  exposing  a  large  surface  to  the  air. 

The  Immunisation  of  the  Animal. — The  animals  chosen 
to  furnish  the  antitoxic  serum  should  be  animals  which 
present  a  distinct  natural  immunity  to  ordinary  doses  of 
the  toxin,  and  should  be  sufficiently  large  to  furnish  large 
quantities  of  the  finished  serum.  Behring  originally 
employed  dogs  and  sheep  ;  Aronson  at  first  preferred  the 
goat ;  but  Roux  introduced  the  horse,  which  is  more  easi- 
ly immunized  than  the  other  animals  mentioned,  and, 
being  large  enough  to  furnish  a  considerable  quantity 
of  serum,  recommends  itself  strongly  for  the  purpose. 

The  animal  chosen  should  be  free  from  tuberculosis 
and  glanders,  as  tested  by  tuberculin  and  mallei n,  but 
need  not  be  expensive.  A  horse  with  a  disabled  foot 
will  answer  well.  Rheumatic  horses  should  be  rejected. 
In  the  beginning  a  small  dose  of  the  toxin — about  i 
c.cm. — should  be  given  hypodermically  to  detect  indi- 
vidual susceptibility.  Horses  vary  much  in  this  particu- 
lar, as  Roux  has  pointed  out.  The  author  found  light- 
colored  horses  to  be  distinctly  more  susceptible  than 
dark-colored  ones. 

If  well  borne,  the  preliminary  injection  is  followed  in 
about  eight  days  by  a  larger  dose,  in  eight  days  more  by 


232  PATHOGENIC  BACTERIA. 

a  still  larger  one,  and  the  increase  is  continued  every  eight 
days  or  so,  according  to  the  condition  of  the  animal, 
until  enormous  quantities — 300  c.cm. — are  introduced  at 
a  time. 

The  toxin  causes  some  local  reaction — at  first  a  dis- 
tinct inflammation,  later  a  painful  edema  and  a  febrile 
reaction.  The  amount  of  local  irritation  is  much  less 
marked  when  the  injections  are  made  slowly ;  and  a 
gravity  apparatus,  which  is  filled  with  the  amount  of 
serum  to  be  injected,  suspended  from  the  ceiling  of  the 
stable  so  that  the  toxin  is  allowed  to  take  its  own  time 
to  enter  the  tissues,  can  be  recommended.  Sometimes 
it  takes  an  hour  to  inject  300  c.cm.  in  this  manner. 

The  amount  of  local  reaction,  edema,  etc.,  the  appetite 
and  general  condition,  the  temperature-curve,  and  the 
stability  of  the  body-weight,  must  all  be  taken  into  con- 
sideration, so  that  the  administration  shall  not  be  too 
rapid  and  the  animal  be  thrown  into  a  condition  of 
cachexia  with  toxic  instead  of  antitoxic  blood. 

One  of  the  principal  things  to  be  avoided  is  haste. 
Too  frequent  or  too  large  dosage  is  almost  certain  to  kill 
the  animal. 

Behring  found  that  mixing  the  toxin  with  trichlorid 
of  iodin  lessened  the  irritant  effect  upon  susceptible 
animals. 

The  suggestion  of  Prof.  Pearson,  that  the  large  doses 
of  toxin  might  with  readiness  be  introduced  into  the 
trachea  when  the  absorption  is  good,  has  been  success- 
fully accomplished  by  the  author.  The  absorption  seems 
to  take  place  without  any  change  in  the  toxin,  and  to  be 
as  rapid  as  from  the  subcutaneous  tissue. 

The  Preparation  of  the  Serum  for  Therapeutic  Pur- 
poses.— When,  because  of  the  tolerance  to  large  quanti- 
ties of  toxin,  the  horse  seems  to  possess  antitoxic  blood, 
a  " twitch"  is  applied  to  the  upper  lip,  the  eyes  are 
blindfolded,  a  small  incision  is  made  through  the  skin,  a 
trocar  thrust  into  the  jugular  vein,  and  the  blood  allowed 
to  flow  through  a  cannulated  tube  into  sterile  bottles.  It 


DIPHTHERIA.  233 

is  allowed  to  coagulate,  and  remains  upon  ice  for  two 
days  or  so,  that  the  clear  serum  may  be  pipetted  off. 
This  serum  is  the  antitoxic  serum.  It  does  not  always 
materialize  according  to  the  desires  of  the  experimenter, 
sometimes  proving  unexpectedly  strong  in  a  short  time, 
sometimes  unexpectedly  weak  after  months  of  patient 
preparation. 

The  strength  of  the  serum  is  expressed  in  what  are 
known  as  immunising  units.  This  denomination  origin- 
ated with  Behring,  whose  original  or  normal  serum  was 
of  such  strength  that  o.  i  c.  cm.  of  it  would  protect  against 
the  ten-times  fatal  dose  of  toxin  when  simultaneously  in- 
jected into  guinea-pigs.  Each  cubic  centimeter  of  this 
normal  serum  he  called  an  immunising  unit.  Later  it 
was  shown  that  the  strength  of  the  serum  could  easily  be 
increased  tenfold,  so  that  o.oi  c.cm.  of  the  serum  would 
protect  the  guinea-pig  against  the  ten-times  fatal  dose. 
Bach  cubic  centimeter  of  this  stronger  serum  was  de- 
scribed as  an  antitoxic  unit,  and  of  course  contained 
ten  immunizing  units.  Still  later  it  was  shown  that 
the  limits  were  by  no  means  reached,  and  he  succeeded 
in  making  serums  as  much  as  three  hundred  times  the 
normal  strength,  each  cubic  centimeter  of  which  con- 
tained 300  immunizing  units  or  30  antitoxic  units. 

The  serums  ordinarily  sold  are  of  three  strengths — 600 
units  in  10  c.cm.,  1000  units  in  10  c.cm.,  and  1500  units 
in  10  c.cm.  The  weaker  strength  is  used  for  immunizing 
healthy  children  and  adults  who  come  in  contact  with 
the  contagium.  The  stronger  serums  are  for  treatment. 
There  is,  of  course,  no  way  of  estimating  the  amount  of 
toxin  in  the  blood  of  a  child  suffering  with  diphtheria, 
and  therefore  no  accurate  method  of  determining  exactly 
how  much  antitoxin  should  be  given.  Khrlich  asserts 
that  less  than  500  units  is  valueless:  10  c.cm.  is  probably 
an  average  dose,  and,  as  the  remedy  seems  harmless,  it  is 
better  to  err  on  the  side  of  too  much  than  on  that  of 
too  little. 

The  largest  collection  of  statistics  upon  the  results  of 


234  PATHOGENIC  BACTERIA, 

antitoxic  treatment  in  diphtheria  in  the  hospitals  of  the 
world  are  probably  those  collected  by  Prof.  Welch,  who, 
excluding  every  possible  error  in  the  calculations,  u  shows 
an  apparent  reduction  of  case-mortality  of  55.8  per  cent." 

One  of  the  most  important  things  in  the  treatment  is 
to  begin  it  early  enough.  Welch's  statistics  show  that 
1115  cases  of  diphtheria  treated  in  the  first  three  days 
of  the  disease  yielded  a  fatality  of  8.5  per  cent.,  whereas 
546  cases  in  which  the  antitoxin  was  first  injected  after 
the  third  day  of  the  disease  yielded  a  fatality  of  27.8  per 
cent. 

After  the  toxin  has  set  up  destructive  organic  lesions 
in  various  organs  and  tissues  of  the  body,  no  amount 
of  neutralization  will  restore  the  integrity  of  the  parts, 
so  that  the  antitoxin  must  fail  in  these  cases. 

The  urticaria  which  sometimes  follows  the  injection 
of  antitoxic  serum  seems  to  bear  a  distinct  relation  to 
the  age  of  the  serum,  fresh  serums  being  more  liable 
to  produce  it  than  those  which  have  been  kept  for  a 
week  or  two. 

The  erythemata  are  probably  in  some  way  associated 
with  the  globulicidal  action  of  the  blood.  Keeping  the 
serum  "  until  it  is  ripe  "  lessens  this  effect.  The  serums 
from  different  horses  probably  vary  much  in  both  their 
irritant  and  globulicidal  properties,  so  that  antitoxins 
prepared  by  mixing  the  serums  from  a  number  of  horses 
are  probably  preferable  to  those  from  single  horses. 

Dried  serums  are  much  less  active  than  fresh  ones. 

For  purposes  of  immunization  smaller  doses  than  those 
used  for  treatment  suffice.  According  to  Biggs,  2  cubic 
centimeters  are  sufficient  to  give  complete  protection. 
The  immunity  that  results  from  the  injection  is  of  a 
month  or  six  weeks'  duration. 


CHAPTER    II 
TETANUS. 

ONE  of  the  most  exquisitely  toxic  bacteria  of  which 
we  have  any  knowledge  is  the  bacillus  discovered  in 
1884  by  Nicolaier,  obtained  in  pure  culture  by  Kitasato 
in  1889,  and  now  universally  recognized  as  the  cause  of 
tetanus.  It  is  a  peculiar  organism,  whose  striking  feature 
is  a  considerable  enlargement  of  one  end,  in  which  a 
bright  round  spore  is  seen  (Fig.  66).  The  bacilli,  which 


FIG.  66. — Bacillus  tetani;    x  looo  (Frankel  and  Pfeiffer). 

are  not  sporiferous,  are  long,  rather  slender,  have  rounded 
ends,  seldom  unite  in  chains  or  pairs,  are  non-motile,  and 
have  no  flagella.  The  bacilli  stain  readily  with  ordi- 
nary aqueous  solution  of  the  anilin  dyes,  and  also  very 
readily  by  Gram's  method. 

The  tetanus  bacillus  is  a  common  saprophytic  organ- 
ism which  can  be  found  in  most  garden-earth,  in  dust, 

235 


236 


PATHOGENIC  BACTERIA. 


in  manure,  and  sometimes  in  the  intestinal  discharges 
of  animals.  It  is  extremely  difficult  to  isolate  and  culti- 
vate, because  it  will  not  grow  where  the  smallest  amount 
of  oxygen  is  present. 

The  method  now  generally  employed  for  the  isolation 
of  this  bacillus  is  that  originated  by  Kitasato,  and  based 
upon  his  observation  that  its  spores  can  resist  high  temper- 


FiG.  67. — Bacillus  tetani :  six-days-  FIG.  68. — Bacillus  tetani :  culture 
old  puncture-culture  in  glucose-gelatin  four  days  old  in  glucose-gelatin  (Fran- 
(Frankel  and  Pfeiffer).  kel  and  Pfeiffer). 

atures.  After  finding  that  the  typical  bacilli  are  present 
in  earth  or  pus,  or  whatever  the  material  to  be  investi- 
gated was,  Kitasato  exposed  a  portion  of  it  for  an  hour 
to  a  temperature  of  80°  C.  By  this  heating  all  the  fully- 
developed  bacteria,  tetanus  as  well  as  the  others,  and  the 


TETANUS. 


237 


great  majority  of  the  spores  except  those  of  tetanus,  were 
destroyed,  and,  as  little  other  than  tetanus  spores  re- 
mained, their  cultivation  was  made  comparatively  easy. 
The  resistance  which  the  tetanus  bacilli  manifest  toward 
heat  is  only  part  of  a  great  general  resisting  power  of 
which  they  are  possessed.  It  is  said  that  they  can  retain 
their  vitality  in  the  dried  condition  for  months.  Stern- 
berg  says  they  can  resist  5  per  cent,  carbolic  solutions' 
for  ten  hours,  but  will  not  grow  after  fifteen  hours'  im- 
mersion. 5  per  cent,  carbolic  acid,  to  which  o.  5  per  cent. 


FIG.  69. — Bacillus  tetani :  five-days-old  colony  upon  gelatin  containing  glucose ; 
x  1000  (Frankel  and  Pfeiffer). 

of  hydrochloric  acid  has  been  added,  destroys  them  in 
two  hours.  They  are  also  destroyed  in  three  hours  by 
i  :  1000  bichlorid-of-mercury  solution  ;  but  when  to  such 
a  solution  0.5  per  cent,  of  hydrochloric  acid  is  added,  its 
activity  is  so  increased  that  the  spores  are  destroyed  in 
thirty  minutes.  The  resistance  to  heat  is  only  within 
certain  limits,  for  exposure  to  passing  steam  for  from 
five  to  eight  minutes  is  certain  to  kill  the  spores. 
The  colonies  of  the  tetanus  bacillus,  when  grown  in 


238  PATHOGENIC  BACTERIA. 

an  atmosphere  of  hydrogen  upon  gelatin  plates,  somewhat 
resemble  those  of  the  well-known  hay  bacillus.  There 
is  a  dense  rather  opaque  central  mass  from  which  a  more 
transparent  zone  is  readily  separable.  The  margins  of 
this  outer  zone  are  made  up  of  a  radiating  fringe  of  pro- 
jecting bacilli  (Fig.  69).  The  liquefaction  that  occurs  is 
much  slower  than  that  caused  by  bacillus  subtilis. 

When  grown  in  gelatin  puncture-cultures  the  develop- 
ment occurs  deep  in  the  puncture,  and  consists  of  mul- 
titudes of  short-pointed  processes  radiating  from  the 
puncture,  somewhat  resembling  a  fir  tree  (Fig.  67). 
Liquefaction  begins  in  the  second  week  and  causes  the 
disappearance  of  the  radiating  processes.  The  liquefac- 
tion spreads  slowly,  but  may  involve  the  entire  mass  of 
gelatin  and  resolve  it  into  a  grayish-white  syrupy  liquid, 
at  the  bottom  of  which  the  bacilli  accumulate.  The 
growth  in  gelatin  containing  glucose  is  much  more  rapid  ; 
that  in  agar-agar  punctures  is  much  slower,  but  similar 
to  the  gelatin  cultures  except  for  the  absence  of  liquefac- 
tion. The  organism  can  also  be  grown  in  bouillon,  and 
attains  its  maximum  development  at  a  temperature  of 
37°  C.  Much  gas  is  given  off  from  the  cultures. 

Cultures  of  the  tetanus  bacillus  in  all  media  give  off 
a  peculiar  characteristic  odor — a  burnt-onion  smell,  with 
a  suggestion  of  putrefaction  about  it. 

The  methods  for  excluding  the  oxygen  from  the  cul- 
tures and  replacing  it  by  hydrogen,  as  well  as  other 
methods  suggested  for  the  cultivation  of  the  strictly 
anaerobic  organisms,  are  given  under  the  appropriate 
heading  (Anaerobic  Cultures),  and  need  not  be  repeated 
here. 

Tetanus  bacilli  exist  in  nature  as  widely  distributed 
saprophytes.  They  are  quite  common  in  the  soil,  and 
the  fact  that  they  are  most  plentiful  in  manured  ground 
has  suggested  that  they  originate  in  the  intestines  of 
horses  and  reach  the  earth  from  their  excrement.  I/e 
Dentu  has,  however,  shown  that  the  tetanus  bacillus  is 
a  common  organism  in  New  Hebrides,  where  there  are  no 


TETANUS.  239 

horses.  In  these  islands  the  natives  poison  their  arrows 
by  dipping  them  into  a  clay  rich  in  tetanus  bacteria. 

The  work  of  Kitasato  has  given  us  a  very  exact 
knowledge  of  the  tetanus  bacillus  and  completely  estab- 
lishes its  specific  nature. 

The  organisms  generally  enter  the  animal  body  through 
a  wound  caused  by  some  implement  which  has  been  in 
contact  with  the  soil,  or  enter  abrasions  from  the  soil 
directly.  Doubtless  many  of  the  wounds  are  so  small 
that  their  existence  is  overlooked,  and  this,  together 
with  the  fact  that  the  period  of  incubation  of  the  dis- 
ease, especially  in  man,  is  of  considerable  duration,  and 
at  times  permits  the  wound  to  heal  before  any  symptoms 
of  intoxication  occur,  serves  to  explain  to  us  at  least  some 
of  the  reported  cases  in  which  no  wound  is  said  to  have 
existed. 

It  would  seem  that  in  some  rare  cases  tetanus  can  occur 
without  the  previous  existence  of  a  wound.  Such  a  case 
has  been  reported  by  Kamen,  who  found  that  the  intes- 
tine of  a  person  dead  of  the  disease  was  rich  in  the 
Bacillus  tetani.  Kamen  is  of  the  opinion  that  the 
bacilli  can  grow  in  the  intestine  and  be  absorbed,  espe- 
cially where  there  are  imperfections  in  the  mucosa.  It 
is  not  impossible,  though  he  does  not  think  it  probable, 
that  the  bacteria  growing  in  the  intestine  could  elaborate 
enough  toxin  to  produce  the  disease  by  absorption. 

All  animals  are  not  alike  susceptible  to  the  disease. 
Men,  horses,  mice,  rabbits,  and  guinea-pigs  are  all  sus- 
ceptible ;  dogs  are  much  less  so.  Most  birds  are  scarcely 
at  all  susceptible  either  to  the  bacilli  or  to  the  poison. 
Amphibians  are  immune,  though  it  is  said  that  frogs 
can  be  made  susceptible  by  elevation  of  their  body- 
temperature. 

When  a  white  mouse  is  inoculated  with  an  almost 
infinitesimal  amount  of  bouillon  or  solid  culture,  or  is 
inoculated  with  garden-earth  containing  the  tetanus 
bacillus,  the  disease  is  almost  certain  to  follow,  the 
first  symptoms  coming  on  in  from  one  to  two  days. 


240  PATHOGENIC  BACTERIA. 

The  mouse  develops  typical  tetanic  convulsions,  which 
begin  first  in  the  neighborhood  of  the  inoculation,  but 
soon  become  general.  Death  follows  sometimes  in  a 
very  few  hours.  In  rabbits  the  period  of  incubation  is 
nearly  two  weeks,  and  in  man  may  be  three  weeks. 

The  conditions  in  the  animal  body  are  not  favorable 
for  the  development  of  the  bacilli,  because  of  the  free 
supply  of  oxygen  contained  in  the  blood,  and  we  find 
that  they  grow  with  great  slowness,  remain  localized  at 
the  seat  of  inoculation,  and  never  enter  the  blood-  or 
lymph-circulation.  Doubtless  most  cases  of  tetanus  are 
cases  of  mixed  infection  in  which  the  bacillus  enters  with 
bacteria,  which  greatly  aid  its  growth  by  using  up  the 
oxygen  in  their  neighborhood.  The  amount  of  poison 
produced  must  be  exceedingly  small  and  its  power  tre- 
mendous, else  so  few  bacilli  growing  under  adverse  con- 
ditions could  not  produce  fatal  toxemia.  The  poison  is 
produced  rapidly,  for  Kitasato  found  that  if  mice  were 
inoculated  at  the  root  of  the  tail,  and  afterward  the  skin 
and  the  subcutaneous  tissues  around  the  inoculation  were 
either  excised  or  burned  out,  this  treatment  would  not 
save  the  animal  unless  the  operation  were  performed 
within  an  hour  after  the  inoculation. 

The  circulating  blood  of  diseased  animals  is  fatal  to 
susceptible  animals  because  of  the  toxin  which  it  con- 
tains ;  and  that  the  urine  is  also  toxic  to  mice  proves  the 
excretion  of  the  toxin  through  the  kidneys. 

From  pure  cultures  of  tetanus  bacilli  grown  in  various 
media,  and  from  the  blood  and  tissues  of  animals  affected 
with  the  disease,  Brieger  has  succeeded  in  separating  two 
poisonous  substances — "tetanin"  and  "  tetano-toxin. " 

The  pathology  of  the  disease  is  of  much  interest  be- 
cause of  its  purely  toxic  nature.  There  is  generally  a 
small  wound  with  a  slight  amount  of  suppuration.  At 
the  autopsy  the  organs  of  the  body  are  normal  in  appear- 
ance, except  the  nervous  system,  which  bears  the  great- 
est insult.  It,  however,  shows  little  else  than  congestion 
either  macroscopically  or  microscopically. 


TETANUS.  241 

An  interesting  fact  contributed  to  our  knowledge  of 
the  disease  has  been  presented  by  Vaillard  and  Rouget, 
who  found  that  if  the  tetanus  bacilli  were  introduced 
into  the  body  freed  from  their  poison,  they  were  unable 
to  produce  any  signs  of  disease  because  of  the  prompt- 
ness with  which  the  phagocytes  took  them  up.  If,  how- 
ever, their  poison  was  not  removed,  or  if  the  body-cells 
were  injured  by  the  simultaneous  introduction  of  lactic 
acid  or  other  chemical  agents,  the  bacilli  would  imme- 
diately begin  to  manufacture  the  toxin  and  produce 
symptoms  of  the  disease. 

The  toxin  is  easily  prepared,  being  readily  soluble  in 
water.  The  most  ready  method  of  preparation  is  to 
grow  the  bacilli  in  bouillon,  keeping  the  culture-medium 
at  a  temperature  of  37°  C.,  and  allowing  it  to  remain  un- 
disturbed for  from  two  to  four  weeks,  by  which  time  it 
will  have  attained  a  toxicity  so  great  that  0.000005  c.cm. 
will  cause  the  death  of  a  mouse.  The  toxin  is  very  rapidly 
destroyed  by  heat,  and  cannot  bear  any  temperature  above 
60-65°  C.  It  is  a^so  decomposed  by  light.  When  pre- 
served in  the  dark  in  a  refrigerator  it  can  be  kept  in- 
definitely. The  best  method  of  keeping  it  is  to  add  0.5 
per  cent,  of  phenol,  and  then  store  it  in  a  cool,  dark  place. 

By  the  gradual  introduction  of  such  a  toxin  into  ani- 
mals Behring  and  Kitasato  have  been  able  to  produce  in 
their  blood  a  distinctly  potent  and  valuable  antitoxic 
substance. 

The  method  for  the  production  of  this  tetanus  anti- 
toxic serum  is  very  much  like  that  for  the  diphtheria 
antitoxic  serum  (q.  v.\  except  that  a  much  longer  time 
is  required  for  its  production,  that  the  doses  of  toxin  are 
of  necessity  smaller  because  its  toxicity  is  greater,  and 
that  trichlorid  of  iodin  or  Gram's  solution  will  probably 
need  to  be  added  to  the  toxin  to  prevent  too  powerful  a 
local  reaction.  Horses,  dogs,  and  goats  may  be  used. 

As  tetanus  cases  are  not  very  common,  and  the  anti- 
toxic serum  when  produced  is  not  very  stable  in  its  prop- 
erties, Tizzoni  and  Cattani  have  successfully  prepared  it 

16 


242  PATHOGENIC  BACTERIA. 

in  a  solid  form,  in  which,  it  is  claimed,  it  can  be  kept 
indefinitely,  shipped  any  distance,  and  used  after  simple 
solution  in  water.  Their  method  is  to  precipitate  the 
antitoxin  from  the  blood  of  immunized  dogs  with  alcohol. 
Numerous  cases  of  the  beneficial  action  of  this  antitoxin 
are  on  record. 

As  Welch  has  pointed  out,  the  antitoxin  of  tetanus  has 
proved  to  be  rather  a  disappointment  in  human  medicine, 
and  also  for  the  treatment  of  large  animals,  such  as  the 
horse.  The  results  following  its  injection,  in  combination 
with  the  sterile  toxin,  into  mice,  guinea-pigs,  and  rabbits 
are  highly  satisfactory,  but  the  amount  needed,  in  pro- 
portion to  the  body-weight,  to  save  the  animal  from  the 
toxin  being  manufactured  in  its  body  by  bacilli  increases 
so  enormously  with  the  day  or  hour  of  the  disease  as  to 
make  the  dosage,  which  increases  millions  of  times  where 
that  of  diphtheria  antitoxin  increases  but  tenfold,  a  matter 
of  difficulty  and  uncertainty.  Nocard  also  calls  atten- 
tion to  the  fact  that  the  existence  of  tetanus  is  unknown 
until  there  is  sufficient  toxemia  to  produce  spasms,  and 
that  therefore  it  is  impossible  to  attack  the  disease  in  its 
inception ;  we  are  obliged  to  meet  it  upon  the  same 
grounds  as  diphtheria  in  the  later  days  of  the  disease — 
a  time  when  it  is  well  known  that  the  chances  of  im- 
provement are  greatly  lessened. 

Of  course,  as  there  is  no  other  remedy  that  combats 
the  disease  at  all,  the  antitoxin  is  one  which,  when  ob- 
tainable, should  always  be  employed. 


CHAPTER    III. 

HYDROPHOBIA,   OR  RABIES. 

No  micro-organism  of  hydrophobia  has  as  yet  been 
discovered,  yet  the  peculiarities  of  the  disease  are  such 
as  to  leave  no  doubt  in  the  mind  of  a  bacteriologist  that 
one  must  exist.  To  find  it  is  now  the  desideratum. 

Although  many  men  have  labored  upon  hydrophobia, 
no  name  is  so  well  known  or  so  justly  honored  as  that 
of  the  great  pioneer  in  bacteriology,  Pasteur.  The  profes- 
sion and  laity  are  alike  familiar  with  his  name  and  work, 
and  although  at  times  the  newspapers  of  our  country 
and  certain  members  of  the  profession  have  opposed  the 
methods  of  treatment  which  he  has  suggested  as  the  re- 
sult of  his  experimentation,  we  cannot  but  feel  that  this 
skepticism  and  opposition  are  due  either  to  ignorance 
of  the  principles  upon  which  Pasteur  reasoned  or  to  a 
culpable  conservatism.  The  most  vehement  opponent 
that  Pasteur  has  in  America  seems  to  disbelieve  the 
existence  of  rabies.  It  is  impossible  to  argue  with  him. 

Hydrophobia,  or  rabies,  is  a  specific  toxemia  to  which 
dogs,  wolves,  skunks,  and  cats  are  highly  susceptible, 
and  which  can,  through  their  saliva,  be  communicated 
to  men,  horses,  cows,  and  other  animals.  The  means 
of  communication  is  almost  invariably  a  bite,  hence  the 
inference  that  the  specific  organism  is  present  in  the 
saliva. 

The  animals  that  are  infected  manifest  no  symptoms 
during  a  varying  incubation-period  in  which  the  wound 
generally  heals  kindly.  This  period  may  last  for  as  long 
a  time  as  twelve  months,  but  in  rare  cases  may  be  only 
some  days.  An  average  duration  of  the  period  of  incu- 
bation might  be  stated  as  about  six  weeks. 

243 


244  PATHOGENIC  BACTERIA. 

As  the  incubation-period  comes  to  an  end  there  is  an 
observable  alteration  in  the  wound,  which  becomes  red- 
dened, sometimes  may  suppurate  a  little,  and  is  painful. 
The  victim,  if  a  man,  is  much  alarmed  and  has  a  sensa- 
tion of  horrible  dread.  The  period  of  dread  passes  into 
one  of  excitement,  which  in  many  cases  amounts  to  a 
wild  delirium  and  ends  in  a  final  stage  of  convulsion  and 
palsy.  The  convulsions  are  tonic,  rarely  clonic,  and 
subsequently  cause  death  by  interfering  with  the  respira- 
tion, as  do  those  of  tetanus  and  strychnia. 

During  the  convulsive  period  much  difficulty  is  experi- 
enced in  swallowing  liquids,  and  it  is  supposed  that  the 
popular  term  "hydrophobia"  arose  from  the  reluctance 
of  the  diseased  to  take  water  because  of  the  inconveni- 
ence and  occasional  spasms  which  the  attempt  causes. 

This  description,  brief  and  imperfect  as  it  is,  will 
illustrate  the  parallelism  existing  between  hydrophobia 
and  tetanus.  In  the  latter  we  observe  the  entrance  of 
infectious  material  through  a  wound,  which,  like  the 
bite  in  hydrophobia,  sometimes  heals,  but  often  suppu- 
rates a  little.  We  see  in  both  affections  an  incubation- 
period  of  varying  duration,  though  in  hydrophobia  it  is 
much  longer  than  in  tetanus,  and  convulsions  of  tonic 
character  causing  death  by  asphyxia. 

It  is  maintained  by  some  that  the  stage  of  excitement 
argues  against  the  specific  nature  of  the  disease,  but 
these  subjective  symptoms  are  like  the  mental  con- 
dition of  tuberculosis,  which  leads  the  patient  to  make  a 
hopeful  prognosis  of  his  case,  and  the  mental  condition 
of  anthrax,  in  which  it  is  stated  that  no  matter  how  dan- 
gerous his  condition  the  patient  is  seldom  much  alarmed 
about  it. 

Pasteur  and  his  co-workers  found  that  in  animals  that 
die  of  rabies  the  salivary  glands,  the  pancreas,  and  the 
nervous  system  contain  the  infection,  and  are  more 
appropriate  for  experimental  purposes  than  the  saliva, 
which  is  invariably  contaminated  with  accidental  patho- 
genic bacteria. 


HYDROPHOBIA,    OR  RABIES.  245 

The  introduction  of  a  fragment  of  the  medulla  ob- 
longata  of  a  dog  dead  of  rabies  beneath  the  dura  mater 
of  a  rabbit  causes  the  development  of  rabies  in  the 
rabbit  in  a  couple  of  weeks.  The  medulla  of  this  rabbit 
introduced  beneath  the  dura  mater  of  a  second  rabbit 
produced  a  more  violent  form  of  the  disease  in  a  shorter 
time,  and  by  frequently  repeated  implantations  Pasteur 
found  that  an  extremely  virulent  material  could  be  ob- 
tained. 

Inasmuch  as  the  toxins  of  diphtheria  and  tetanus 
circulate  in  the  blood,  and  not  infrequently  saturate 
the  nervous  systems  of  animals  affected,  it  might  be 
concluded  that  the  material  with  which  Pasteur  worked 
was  a  toxin.  This  is  readily  disproven,  however,  not 
only  by  the  fact  that  the  toxin  would  weaken  instead  of 
strengthen  by  the  method  of  transfer  from  animal  to 
animal,  it  not  being  a  vital  entity,  but  also  by  the  dis- 
covery that  when  an  emulsion  of  the  nervous  system  of 
an  affected  animal  is  filtered  through  porcelain,  or  when 
it  is  heated  for  a  few  moments  to  100°  C.,  or  exposed 
for  a  considerable  time  to  a  temperature  of  75°  or  80°  C., 
its  virulence  is  entirely  lost.  This  would  seem  to  prove 
that  that  which  is  in  the  nervous  system  and  communi- 
cates the  disease  is  a  living,  active  body — a  parasite,  and 
in  all  probability  a  bacterium.  However,  all  endeavors 
to  discover,  isolate,  or  cultivate  this  organism  have  failed. 

Pasteur  noted  that  the  virulence  of  the  poison  was  less 
in  animals  that  had  been  dead  for  some  time  than  in 
the  nervous  systems  of  those  just  killed,  and  by  experi- 
mentation showed  that  when  the  nervous  system  was 
dried  in  a  sterile  atmosphere  the  virulence  was  attenu- 
ated in  proportion  to  the  length  of  time  it  had  been  dry. 
This  attenuation  of  virulence  of  course  suggested  to 
Pasteur  the  idea  of  a  protective  vaccination,  and  by  in- 
oculating a  dog  with  much  attenuated,  then  with  less 
attenuated,  then  with  moderately  strong,  and  finally  with 
strong,  virus,  the  dog  developed  an  immunity  which 
enabled  it  to  resist  the  infection  of  an  amount  of  viru- 


246  PATHOGENIC  BACTERIA. 

lent  material  that  would  certainly  kill  an  unprotected 
animal. 

It  is  remarkable  that  this  thought,  which  was  a  theory 
based  upon  a  broad  knowledge,  but  experience  with 
comparatively  few  bacteria,  should  every  day  find  more 
and  more  grounds  for  confirmation  as  our  knowledge 
of  immunity,  of  toxins,  and  of  antitoxins  progresses. 
What  Pasteur  did  with  rabies  is  what  we  now  do  in 
producing  the  antitoxin  of  diphtheria — i.  e.  gradually 
accommodate  the  animal  to  the  poison  until  its  body-cells 
are  able  to  neutralize  or  resist  it.  As  the  poison  cannot 
be  secured  outside  of  the  body  because  the  bacilli,  micro- 
cocci,  or  whatever  they  may  be  cannot  be  secured  outside 
of  the  body,  he  does  what  Behring  originally  did  in  diph- 
theria— introduces  attenuated  poison-producers — bacilli 
crippled  by  heat  or  drying,  and  capable  of  producing  only 
a  little  poison — accustoms  the  animal  to  these,  and  then  to 
stronger  and  stronger  ones  until  immunity  is  established. 

The  genius  of  Pasteur  did  not  cease  with  the  produc- 
tion of  immunity,  but,  we  rejoice  to  add,  extended  to  the 
kindred  subject  of  therapy,  and  has  now  given  us  a  cure 
for  hydrophobia. 

For  the  production  of  a  cure  in  infected  cases  very 
much  the  same  treatment  is  followed  as  has  been  de- 
scribed for  the  production  of  immunity.  The  patient 
must  come  under  observation  early.  The  treatment  con- 
sists of  the  subcutaneous  injection  of  about  2  grams  of 
an  emulsion  of  a  rabbit's  spinal  cord  which  had  been 
dried  for  from  seven  to  ten  days.  This  beginning  dose 
is  not  increased  in  size,  but  each  day  the  emulsion  used 
is  from  a  cord  which  has  not  been  dried  so  long,  until, 
when  the  twenty-fifth  day  of  treatment  is  reached,  the 
patient  receives  2  grams  of  emulsion  of  spinal  cord  dried 
only  three  days,  and  is  considered  immune  or  cured. 

It  will  be  observed  that  this  treatment  is  really  no 
more  than  the  immunization  of  the  individual  during  the 
incubation  stadium,  and  the  generation  of  a  vital  force — 
shall  we  call  it  an  antitoxin  ? — in  the  blood  of  the  animal 


HYDROPHOBIA,    OR  RABIES.  247 

in  advance  of  the  time  when  the  organism  is  expected  to 
saturate  the  body  with  its  toxic  products. 

This,  in  brief,  is  the  theory  and  practice  of  Pasteur's 
system  of  treating  hydrophobia.  It  is  exactly  in  keeping 
with  the  ideas  of  the  present,  and  is  most  extraordinary 
in  its  reasonings  and  details  when  we  remember  that  the 
first  application  of  the  method  to  human  medicine  was 
made  October  26,  1885,  nearly  ten  years  before  the  time 
we  began  to  understand  the  production  and  use  of  anti- 
toxins. 


CHAPTER    IV. 
SYMPTOMATIC  ANTHRAX. 

k  l  SYMPTOMATIC  ANTHRAX,  ' '  charbon  symptomatique, 
Rauschbrand,  "quarter- evil,"  and  "black-leg"  are  the 
various  names  applied  to  a  peculiar  disease  of  cattle  com- 
mon during  the  summer  season  in  the  Bavarian  Alps, 
Baden,  Schleswig-Holstein,  and  some  parts  of  the  United 
States,  characterized  by  the  occurrence  of  irregular,  em- 
physematous,  crepitating  subcutaneous  pustules.  Dis- 
eased areas  are  also  found  in  the  muscles,  and  are  most 
common  over  the  quarters,  hence  the  name  ' '  quarter- 
evil."  When  incised  the  affected  tissues  have  a  dark 
color  and  contain  a  dark,  bloody  serum. 

The  micro-organismal  nature  of  the  disease  had  been 
suspected  from  an  early  date,  but  until  the  work  of 
Faser  and  Bellinger  the  disease  was  confounded  with 
anthrax.  Still  later,  Arloing,  Thomas,  Cornevin,  and 
Kitasato  studied  the  disease,  and  succeeded  in  demon- 
strating the  specific  micro-organism,  which  Kitasato 
successfuly  cultivated  upon  artificial  media. 

The  bacillus  which  the  results  of  these  labors  brought 
to  light  is  a  rather  large  individual  (3-5  /j.  in  length, 
0.5-0.6  fj-  in  breadth)  with  rounded  ends.  The  bacilli 
are  occasionally  united  in  twos,  but  are  never  united  in 
long  chains  (Fig.  70).  They  are  actively  motile  (Thoinot 
and  Masselin  say  scarcely  at  all  motile)  when  examined 
in  the  hanging  drop,  but  after  a  short  time,  perhaps 
because  of  the  exposure  to  the  oxygen  required  in  the 
hanging-drop  preparation,  the  movement  is  lost  and  the 
bacilli  die.  When  stained  by  L,6ffler's  method  a  con- 
siderable number  of  flagella  can  be  demonstrated.  Large 

248 


SYMPTOM  A  TIC  ANTHRAX.  249 

•oval  spores  are  found ;  by  their  presence  they  distort  the 
bacilli  in  which  they  occur,  causing  them  to  assume  a 
spindle  shape  (clostridium),  or,  when  two  are  united  and 
a  spore  occupies  one  of  them,  a  drumstick  shape.  In- 


FIG.  70. — Bacillus  of  symptomatic  anthrax,  containing  spores,  from   an  agar- 
agar  culture;    x  1000  (Frankel  and  Pfeiffer). 


volution-forms  are  exceedingly  common  in  old  cul- 
tures, and  are  of  enormous  size  and  of  granular  appear- 
ance. 

The  bacillus  can  be  stained  with  the  ordinary  aqueous 
solutions  of  the  anilin  dyes,  but  will  not  retain  the  color 
by  Gram's  method  or  Weigert's  fibrin  method.  It  can 
be  colored  in  sections  of  tissue  with  Loffler's  solution, 
and  can  be  observed  in  the  blood  without  staining  shortly 
after  death. 

The  spores,  which  can  be  stained  by  ordinary  methods, 
are  quite  resistant  to  the  action  of  heat  and  disinfect- 
ants, and  withstand  the  effects  of  drying  for  a  consider- 
able length  of  time. 

The  bacillus  of  symptomatic  anthrax  (Fig.  71)  is  a 
strictly  anaerobic,  parasitic  bacterium.  It  grows  at  tem- 
peratures above  18°  C.,  but  best  at  37°  C. 


250 


PATHOGENIC  BACTERIA. 


The    artificial 
Kitasato   is   not   more 


cultivation  which  was  achieved  by 
difficult  than  that  of  other  an- 
aerobic organisms.  In  gelatin 
containing  i  to  2  per  cent,  of 
glucose  or  5  per  cent,  of  gly- 
cerin the  organism  develops 
quite  well,  the  exact  appearance 
depending  somewhat  upon  the 
method  by  which  it  was  planted. 
If  the  bacteria  are  dispersed 
through  the  culture -medium, 
the  little  colonies  will  appear 
in  the  lower  parts  of  the  tube  as 
nearly  spherical  or  slightly  irreg- 
ular, clouded,  liquefied  areas  con- 
taining bubbles  of  gas.  If,  on 
the  other  hand,  the  inoculation 
is  made  by  a  deep  puncture,  a 
stocking  -  shaped  liquefaction 
forms  along  the  whole  lower 
part  of  the  puncture,  leads  to 
considerable  gas-production,  and 
finally  causes  the  liquefaction  of 
all  the  gelatin  except  a  thin 
superficial  stratum.  A  peculiar 
acid  odor  is  given  off  by  the 
cultures. 

When  the  bacteria  grow  anaerobically  in  Ksmarch 
tubes,  the  colonies  are  irregularly  club-shaped  or  spheri- 
cal, with  a  tangled  mass  of  delicate  projecting  filaments 
visible  upon  microscopic  examination. 

In  agar-agar  the  development  is  similar  to  that  in 
gelatin.  The  gas-production  is  marked,  the  liquefaction 
of  course  absent,  and  the  same  acid  odor  pronounced. 

The  bacillus  also  develops  quite  well  in  bouillon,  the 
bacillary  masses  sinking  to  the  bottom  in  the  form  of 
whitish  flakes,  while  the  gas-bubbles  collect  at  the  top. 
In  this  medium  the  virulence  is  unfortunately  soon  lost. 


FIG.  71. — Bacillus  of  symp- 
tomatic anthrax  :  four-clays-old 
culture  in  glucose-gelatin  (Fran- 
kel  and  Pfeififer). 


SYMPTOMATIC  ANTHRAX.  251 

Milk  also  seems  to  be  a  favorable  culture-medium. 
The  development  of  the  bacilli  is  unaccompanied  by 
coagulation. 

The  virulence  of  the  organism  is  soon  lost  in  all 
culture-media,  but  it  is  said  that  the  virulence  of  the 
culture  can  be  much  increased  by  the  addition  to  it  of 
20  per  cent,  of  lactic  acid. 

When  susceptible  animals  are  inoculated  with  a  minute 
portion  of  a  pure  culture  in  a  little  subcutaneous  pocket, 
such  as  is  described  in  connection  with  tetanus  and 
malignant  edema,  the  bacilli  proceed  to  grow,  pro- 
duce the  well-known  affection,  and  lead  to  a  certainly 
fatal  outcome.  Cows  seem  to  be  the  most  susceptible 
animals,  especially  those  between  six  months  and  four 
years  old ;  sheep  and  goats  are  also  sometimes  affected. 
Curiously  enough,  animals  that  are  immune  to  malig- 
nant edema  are  seemingly  more  susceptible  to  Rausch- 
brand.  Of  the  laboratory  animals,  the  guinea-pig  is 
most  susceptible ;  swine,  dogs,  and  rabbits  are  very 
slightly  susceptible ;  horses,  goats,  and  birds  are  im- 
mune. 

The  virulence  of  the  bacillus  is  capable  of  ready 
attenuation  by  exposure  to  heat,  by  previous  exposure 
of  its  spores  to  heat,  or  by  drying  combined  with  ex- 
posure to  increased  temperature.  The  inoculation  of 
animals  with  the  attenuated  bacilli  causes  a  very  mild 
affection,  followed  by  complete  immunity  to  the  viru- 
lent organisms.  Upon  this  principle  the  "  protective 
vaccination"  is  based.  Kitt  has,  however,  shown  that 
almost  the  same  method  as  that  employed  by  Pasteur 
for  vaccination  against  rabies  may  be  employed  against 
this  bacillus,  and  that  when  muscular  tissue  from  an 
animal  dead  of  the  disease  is  dried  at  a  temperature  of 
32-35°  C.,  and  then  exposed  for  six  hours  to  a  tempe- 
rature of  85-90°  C.,  and  a  second  portion  is  exposed  in 
the  same  manner  to  a  temperature  of  100-104°  C.,  an 
emulsion  of  this  tissue  in  distilled  water,  salt-solution, 
or  bouillon,  injected  into  the  animals  to  be  protected,  will 


252  PATHOGENIC  BACTERIA. 

act  in  a  manner  resembling  the  pulverized  spinal  cords 
of  the  rabbits  used  in  rabies,  and  give  an  almost  per- 
fect immunity.  Roux  and  Chamberland  have  found  that 
filtered  cultures  can  also  produce  immunity  when  properly 
introduced  into  animals. 

The  immunity  to  symptomatic  anthrax  seems,  how- 
ever, to  be  one  of  degree,  for  Arloing,  Cornevin,  and 
Thomas  found  that  when  the  bacillus  was  introduced 
into  the  animal  body  simultaneously  with  a  20  per  cent. 
solution  of  lactic  acid,  either  the  virulence  of  the  bacil- 
lus or  the  resistance  of  the  tissues  was  so  changed  that 
natural  immunity  was  destroyed  and  the  bacteria  allowed 
to  develop  and  produce  the  disease.  Roger  found  also 
that  refractory  animals,  like  the  rabbit,  mouse,  pigeon, 
and  chicken,  could  be  made  susceptible  by  the  combined 
injection  of  the  Rauschbrand  bouillon,  the  Bacillus  pro- 
digiosus,  Proteus  vulgaris,  and  other  harmless  organisms. 

When  the  guinea-pig  is  inoculated  with  the  bacillus  of 
symptomatic  anthrax,  it  dies  in  from  twenty-four  to 
thirty-six  hours.  The  post-mortem  examination  shows 
a  bloody  serum  at  the  point  of  inoculation,  and  the  mus- 
cles are  dark  red  or  black,  like  those  of  the  ' '  black-leg ' ' 
of  cattle.  No  changes  are  apparent  in  the  internal  organs. 
The  bacilli  are  at  first  found  near  the  point  of  inocula- 
tion in  the  inflammatory  exudations  only,  but  soon  after 
death,  being  motile,  they  spread  to  all  parts  of  the  body. 

The  peculiarities  of  symptomatic  anthrax  point  to  the 
entrance  of  the  bacteria  into  the  animal  body  through 
wounds,  but  the  occurrence  of  epidemics  at  certain  geo- 
graphical points,  known  technically  as  * '  Rauschbrand 
stations,"  suggests  that  infection  may  also  take  place 
through  the  respiratory  and  alimentary  tracts. 

At  first  thought,  as  Frankel  points  out,  one  might 
imagine  that  an  animal  dead  of  quarter-evil  and  the  dis- 
charges from  its  body  might  be  harmless,  as  compared, 
for  example,  with  the  cadavers  and  discharges  of  anthrax, 
because  of  the  purely  anaerobic  method  of  the  growth  of 
the  bacillus  of  symptomatic  anthrax  and  the  rapidity  of  its 


SYMPTOMATIC  ANTHRAX.  253 

death  in  the  presence  of  oxygen.  This  is,  however,  un- 
true, for  the  rapid  development  of  a  permanent  form  in 
the  resisting  spores  of  the  bacillus  makes  the  pollution 
of  the  soil  exceedingly  dangerous  for  cows  who  subse- 
quently browse  upon  it.  That  the  spores  are  of  great 
vitality  is  shown  by  the  well-known  laboratory  method 
of  keeping  them  on  hand  for  experimental  purposes,  dried 
in  the  muscular  tissue  of  a  diseased  animal. 

Every  precaution  should  be  exerted  to  have  the  affected 
animals  isolated,  and  their  cadavers  disinfected  and  de- 
stroyed or  buried  in  such  a  manner  that  subsequent 
infection  is  impossible. 

Statistical  results  of  Guillod  and  Simon,  based  upon 
3500  protective  inoculations,  show  a  distinct  reduction 
of  the  death-rate  from  5-20  per  cent,  in  unprotected 
animals  to  0.5-2  per  cent,  in  protected  animals. 


CHAPTER    V. 
TYPHOID  FEVER. 


THE  bacillus  of  typhoid  fever  (Fig.  72)  was  discovered 
by  Eberth  in  1880,  and  was  first  secured  in  pure  culture 


FIG.  72. — Bacillus  typhi,  from  a  twenty-four-hours-old    agar-agar  culture ;    x 

650  (Heim). 

from  the  spleen  and  affected  lymphatic  glands  by  Gaff  ky 
four  years  later. 

The  organism  is  a  small,  short  bacillus  about  1-3  n 
(2-4 1>.  Chantemesse,  Widal)  in  length  and  o.  5-0. 8  P.  broad 
(Sternberg).  The  ends  are  rounded,  and  it  is  rather  ex- 
ceptional for  the  bacilli  to  be  united  in  chains,  though 
this  arrangement  is  common  in  potato  cultures.  The 
size  and  morphology  vary  distinctly  with  the  nature  of 
the  culture-medium  and  the  age  of  the  culture.  Thoinot 
and  Masselin  in  describing  these  morphological  peculi- 
arities mention  that  when  grown  in  bouillon  it  is  a  very 
slender  bacillus ;  in  milk  it  is  a  large  bacillus ;  upon 
agar-agar  and  potato  it  is  very  thick  and  short ;  and  in 
old  gelatin  cultures  it  forms  very  long  filaments. 

254 


TYPHOID  FEVER. 


255 


The  organisms  are  actively  motile,  the  motility  prob- 
ably being  caused  by  the  numerous  flagella  with  which 


FIG.  73.  — Bacillus  coli  communis,  from  an  agar-agar  culture;  x   1000  (Itzerott 

and  Niemann). 

the   bacilli   are   provided.      The   flagella   stain  well   by 
Ivoffler's  method,   and,   as  they  are  numerous  (eighteen 


FIG.  74.— Bacillus  typhi,  from  an  agar-agar  culture  six  hours  old,  showing  the 
flagella  stained  by  Lofflers  method;  x   1000  (Frankel  and  PfeifTer). 

to  twenty)  and  readily  demonstrable,  the  typhoid  bacillus 
is  the  favorite  subject  for  their  study. 


256  PATHOGENIC  BACTERIA. 

The  organism  stains  quite  well  by  the  ordinary  meth- 
ods, but  loses  the  color  entirely  when  stained  by  Gram's 
method.  Its  peculiarity  of  staining  is  the  readiness  with 
which  the  bacillus  gives  up  its  color  in  the  presence  of 
solvents,  so  that  it  is  particularly  difficult  to  stain  it  in 
tissue. 

When  sections  are  to  be  stained  the  best  method  is  to 
allow  the  tissue  to  remain  in  Loffler's  alkaline  methylene 
blue  for  from  fifteen  minutes  to  twenty-four  hours,  then 
wash  in  water,  dehydrate  rapidly  in  alcohol,  clear  up  in 
xylol,  and  mount  in  Canada  balsam.  Ziehl's  method 
also  gives  good  results.  The  sections  are  stained  for  fif- 
teen minutes  in  a  solution  of  distilled  water  100,  fuch- 
sin  i,  and  phenol  5.  After  staining  they  are  washed  in 
distilled  water  containing  i  per  cent,  of  acetic  acid, 
dehydrated  in  alcohol,  cleared,  and  mounted.  In  such 
preparations  the  bacilli  may  be  found  in  little  groups, 
which  are  easily  discovered,  under  a  low  power  of 
the  microscope,  as  bluish  specks,  and  readily  resolved 
into  bacilli  with  the  high  power  of  the  oil-immersion 
lens. 

In  bacilli  stained  by  this  alkaline  methylene-blue  solu- 
tion dark-colored  dots  may  sometimes  be  observed  near 
the  ends  of  the  rods.  These  dots  were  at  first  regarded 
as  spores,  but  are  now  denominated  polar  granules,  and 
are  thought  to  be  of  no  importance. 

The  typhoid  bacillus  is  both  saprophytic  and  parasitic. 
It  finds  abundant  conditions  in  nature  for  its  growth  and 
development,  and,  enjoying  strong  resisting  powers,  can 
accommodate  itself  to  environment  much  better  than  the 
majority  of  pathogenic  bacteria,  and  can  be  found  in 
water,  air,  soiled  clothing,  dust,  sewage,  milk,  etc.  con- 
taminated directly  or  indirectly  by  the  intestinal  dis- 
charges of  diseased  persons. 

The  bacillus  is  also  occasionally  present  upon  green 
vegetables  sprinkled  with  water  containing  it,  and  an 
epidemic  is  reported  in  which  the  infection  was  traced  to 
oysters  from  a  certain  place  where  the  water  was  infected 


TYPHOID  FEVER.  257 

through  sewage.  The  bacillus  probably  enters  milk  oc- 
casionally in  water  used  to  dilute  it. 

The  resistant  powers  of  the  organisms  have  already 
been  described  as  great.  They  can  grow  well  at  the 
room-temperature.  The  thermal  death-point  is  given  by 
Sternberg  as  60°  C.  The  bacilli  can,  according  to  Klem- 
perer  and  Levy,  remain  vital  for  three  months  in  distilled 
water,  though  in  ordinary  water  the  commoner  and  more 
vigorous  saprophytes  outgrow  them  and  cause  their  dis- 
appearance in  a  few  days.  When  buried  in  the  upper 
layers  of  the  soil  the  bacilli  retain  their  vitality  for  nearly 
six  months.  Cold  has  no  effect  upon  the  typhoid  bacilli, 
for  freezing  and  thawing  several  times  are  without  injury 
to  them.  They  have  been  found  to  remain  alive  upon 
linen  for  from  sixty  to  seventy-two  days,  and  upon  buck- 
skin for  from  eighty  to  eighty-five  days.  Sternberg  has 
succeeded  in  keeping  hermetically-sealed  bouillon  cul- 
tures alive  for  more  than  a  year.  In  the  presence  of 
chemical  agents  the  bacillus  is  also  able  to  retain  its 
vitality,  o.  i  to  o.  2  per  cent,  of  carbolic  acid  added  to  the 
culture-media  being  without  effect  upon  its  growth.  At 
one  time  the  tolerance  to  carbolic  acid  was  thought  to  be 
characteristic,  but  it  is  now  known  to  be  shared  by  other 
bacteria. 

Cultures  of  the  typhoid  bacillus  may  be  obtained,  but 
with  difficulty,  from  the  alvine  discharges  of  typhoid 
patients.  In  examining  this  material,  however,  it  must 
be  remembered  that  the  bacilli  are  certain  to  be  present 
only  in  the  second  and  third  weeks. 

As  numerous  saprophytic  bacteria  are  present  in  the 
feces,  the  resistance  which  the  typhoid  bacillus  exhibits 
to  carbolic  acid  can  be  made  use  of  in  obtaining  the  pure 
culture.  To  each  of  several  tubes  of  melted  gelatin  0.05 
per  cent,  of  carbolic  acid  is  added.  This  addition  is  most 
easily  calculated  by  supposing  the  average  amount  of 
gelatin  contained  in  a  tube  to  be  10  c.cm.  To  the  aver- 
age tube  y1^-  c.  cm.  of  a  5  per  cent,  solution  of  carbolic  acid 
is  added,  and  gives  very  nearly  the  desired  quantity.  A 

17 


258 


PATHOGENIC  BACTERIA. 


minute  portion  of  the  feces  is  broken  up  with  a  platinum 
loop  and  stirred  in  the  tube  of  melted  gelatin  ;  a  drop 
from  this  dilution  is  transferred  to  the  second  tube,  a 
drop  from  it  to  a  third,  and  then  the  contents  of  each 
tube  are  poured  upon  a  sterile  plate  or  into  a  Petri  dish, 


FIG.  75. — Bacillus  typhi   abdominalis :   superficial  colony  two  days  old,  as 
seen  upon  the  surface  of  a  gelatin  plate;    x  20  (Heim). 


FIG.  76. — Bacillus  coli  communis  :  superficial  colony  two  days  old  upon  a 
gelatin  plate;    x  21  (Heim). 

or  rolled,  according  to  Esmarch's  plan,  in  the  manner 
already  described.  The  carbolic  acid  present  in  these 
cases  prevents  the  great  mass  of  saprophytes  from  de- 
veloping, but  allows  the  perfect  development  of  the 


TYPHOID  FEVER.  259 

typhoid  bacillus  (Fig.  75)  and  its  near  congener,  the 
Bacillus  coli  communis  (Fig.  76). 

The  colonies  that  develop  upon  such  gelatin  plate- 
cultures  are  seen  under  the  microscope  to  be  brownish- 
yellow  in  color,  spindle-shaped,  and  sharply  circum- 
scribed. When  superficial  they  are  larger  and  form  a 
bluish  iridescent  layer  with  notched  edges.  The  centre 
of  the  superficial  colonies  is  the  only  portion  which 
shows  the  yellowish-brown  color.  The  margins  of  the 
colony  appear  somewhat  reticulated.  The  gelatin  is  not 
liquefied. 

Unfortunately,  the  appearances  of  the  colonies  of  the 
Bacillus  typhi  and  the  Bacillus  coli  communis  are  iden- 
tical, and  make  it  next  to  impossible  to  select  a  single 
colony  of  either  with  any  certainty.  The  only  solution 
of  the  problem  is  to  transfer  a  large  number  of  colonies 
to  some  culture-medium  in  which  a  characteristic  of  one  or 
the  other  species  is  manifested,  and  then  study  the  growth. 

When  transferred  to  gelatin  puncture-cultures  the 
bacilli  develop  along  the  entire  track  of  the  wire,  with 
the  formation  of  minute  confluent  spherical  colonies.  A 
small  thin  whitish  layer  develops  upon  the  surface  near 
the  centre.  The  gelatin  is  not.  liquefied,  but  sometimes 
is  slightly  clouded  in  the  neighborhood  of  the  growth. 
The  growth  upon  the  surface  of  obliquely  solidified  gela- 
tin, agar-agar,  or  blood-serum  is  not  very  luxuriant.  It 
forms  a  thin,  moist,  translucent,  non-characteristic  band 
with  smooth  edges. 

Upon  potato  a  characteristic  growth  takes  place.  When 
the  potato  is  inoculated  and  stood  in  the  incubating  oven, 
no  growth  can  be  detected  at  the  end  of  the  second 
day,  unless  the  observer  be  skilled  and  the  examina- 
tion thorough.  If,  however,  the  medium  be  touched 
with  a  platinum  wire,  it  is  discovered  that  its  entire  sur- 
face is  covered  with  a  rather  thick,  invisible  layer  of  a 
sticky  vegetation  which  the  microscope  shows  to  be  made 
up  of  bacilli.  No  other  bacillus  gives  the  same  kind  of 
growth  upon  potato.  Unfortunately,  it  is  not  constant, 


26o  PATHOGENIC  BACTERIA. 

for  occasionally  there  will  be  encountered  a  typhoid 
bacillus  which  will  show  a  distinct  yellowish  or  brown- 
ish color.  The  typical  growth  seems  to  take  place  only 
when  the  reaction  of  the  potato  is  acid. 

In  bouillon  the  only  change  produced  by  the  growth 
of  the  bacillus  is  a  diffuse  cloudiness. 

In  milk  a  slight  and  slow  acidity  is  produced.  The 
growth  in  milk  is  not  accompanied  by  coagulation. 

The  chief  hindrance  to  the  ready  isolation  of  the 
typhoid  bacillus  is  the  closely-allied  species  described 
by  Escherich  as  the  Bacterium  coli  commune,  by  Em- 
merich as  the  Bacillus  Neapolitanus,  and  now  known 
as  the  Bacillus  coli  communis.  This  organism,  being 
habitually  present  in  the  intestine,  exists  there  in  ty- 
phoid fever,  and  adds  no  little  complication  to  the 
bacteriological  diagnosis  by  responding  in  exactly  the 
same  manner  as  the  typhoid  bacillus  to  the  action  of 
carbolic  acid,  by  having  colonies  almost  exactly  like 
those  of  typhoid,  by  growing  in  exactly  the  same  man- 
ner upon  gelatin,  agar-agar,  and  blood-serum,  by  cloud- 
ing bouillon  in  the  same  way,  by  being  of  exactly  the 
same  shape  and  size,  by  having  flagella,  by  sometimes 
being  motile,  and,  in  fact,  by  so  many  pronounced  simi- 
larities as  to  warrant  the  assertion  of  many  that  it  and 
the  typhoid  bacillus  are  identical. 

At  the  present  time  we  are  in  more  or  less  of  a  quan- 
dary about  this  extraordinary  resemblance,  but  base  our 
differentiation  of  the  species  upon  certain  constant, 
slight,  but  distinct  differences. 

The  Bacillus  coli  communis  grows  differently  upon 
acid  potato,  producing  a  smeary,  elevated,  circumscribed, 
brownish  layer  which  bears  a  resemblance  to  the  growth 
of  the  typhoid  bacillus  upon  alkaline  or  neutral  potato. 
This  bacterium,  in  addition  to  a  more  pronounced  acid- 
production  in  milk,  causes  prompt  coagulation,  which 
the  typhoid  bacillus  does  not. 

When  the  colon  bacillus  is  planted  in  gelatin  or  agar- 
agar  containing  a  small  amount  of  glucose,  a  beautiful 


TYPHOID  FEVER.  261 

gas-production  is  developed,  which  is  unknown  to  the 
typhoid  bacillus. 

Finally,  the  typhoid  bacillus  does  not  produce  indol, 
but  the  addition  of  potassium  nitrite  and  sulphuric  acid 
to  bouillon  cultures  of  the  colon  bacillus  invariably  brings 
about  the  rose  color  which  characterizes  this  product. 

Not  only  are  the  morphological  and  vegetative  similar- 
ities of  these  organisms  great,  but  their  pathogeny  bears 
many  points  of  resemblance.  The  open  lymphatics  and 
vessels  of  the  intestinal  ulcers  of  typhoid  favor  the  ab- 
sorption of  the  bacteria  in  the  digestive  tract,  and  the 
colon  bacillus  enters  the  blood  no  longer  to  be  a  sapro- 
phyte, but  now  to  be  a  virulent  pus-producer,  and  in 
many  cases  of  typhoid  we  find  suppurations  and  other 
milder  inflammations  due  to  this  microbe.  This  is  also 
a  stumbling-block,  for  the  typhoid  bacillus  when  dis- 
tributed through  the  blood  may  act  in  exactly  the  same 
manner. 

The  typhoid  bacillus  may  enter  the  body,  at  times, 
through  dust  (Klemperer  and  Levy),  but  no  doubt,  in  the 
great  majority  of  cases,  enters  the  digestive  tract  at  once 
through  the  mouth.  It  may  possibly  enter  through  the 
rectum  at  times,  as  illustrated  by  the  mention  which 
Eichhorst  makes  of  the  infection  of  soldiers  in  military 
barracks  through  the  wearing  of  drawers  previously  worn 
by  comrades  who  had  suffered  from  typhoid. 

When  ingested  the  resisting  power  of  the  bacillus  per- 
mits it  to  pass  uninjured  through  the  acid  secretions  of 
the  stomach  and  to  enter  the  intestine,  where  the  chief 
local  disturbances  are  set  up. 

The  bacilli  enter  the  solitary  glands  and  Peyer's  patches, 
and  multiply  slowly  during  the  one  to  three  weeks  of  the 
incubation  of  the  disease.  The  immediate  result  of  their 
residence  in  these  lymphatic  structures  is  increase  in  the 
number  of  cells,  and  ultimately  the  necrosis  and  slough- 
ing which  cause  the  typical  post-mortem  lesion.  From 
the  intestinal  lymphatics  the  bacilli  pass,  in  all  probabil- 
ity, to  the  mesenteric  glands,  which  become  enlarged  and 


262  PATHOGENIC  BACTERIA. 

softened,  and  finally  extend  to  the  spleen  and  liver,  and 
sometimes  to  the  kidneys.  The  growth  of  the  bacilli 
in  the  kidneys  causes  the  albuminuria  of  the  disease. 
Sometimes  under  these  conditions  the  bacilli  can  be  found 
in  the  urine.  Occasionally  the  bacilli  succeed  in  entering 
the  general  circulation,  and,  finding  a  lodgement  at  some 
remote  part  of  the  body,  set  up  local  inflammatory  pro- 
cesses sometimes  terminating  in  suppuration. 

The  bacilli  can  be  found  in  the  intestinal  lesions,  in 
the  mesenteric  glands,  in  the  spleen,  in  the  liver,  in  the 
kidneys,  and  in  any  local  lesions  which  may  be  present. 
Their  scattered  distribution  and  their  occurrence  in 
minute  clumps  have  already  been  alluded  to.  They 
should  always  be  sought  for  at  first  with  a  low  power 
of  the  microscope. 

Ordinarily  no  bacilli  can  be  found  in  the  blood,  but 
it  has  been  shown  that  the  blood  in  the  roseolse  some- 
times contains  them,  so  that  the  eruption  may  be  regarded 
as  one  of  the  local  irritative  manifestations  of  the  bacillus. 

The  amount  of  local  disturbance,  in  proportion  to  the 
constitutional  disturbance,  is,  in  the  majority  of  cases, 
slight,  and  almost  always  partakes  of  a  necrotic  charac- 
ter, which  suggests  that  in  typhoid  we  have  to  do  with  a 
toxic  bacterium  whose  disease-producing  capacity  resides 
in  the  elaboration  of  a  toxic  substance.  This,  indeed, 
is  true,  for  Brieger  and  Frankel  have  separated  from 
bouillon  cultures  a  toxalbumin  which  seems  to  be  the 
specific  poison.  Klemperer  and  L,evy  also  point  out 
further  clinical  proof  in  certain  exceptional  cases  dying 
with  the  typical  picture  of  typhoid,  yet  without  charac- 
teristic post-rnortem  lesions,  the  only  confirmation  of  the 
diagnosis  being  the  discovery  of  the  bacilli  in  the  spleen. 

As  the  discovery  of  the  bacilli  in  the  spleen,  and  espe- 
cially the  securing  of  a  pure  culture  of  the  bacilli  from 
the  spleen,  are  sometimes  attended  with  considerable  dif- 
ficulty because  of  the  dissemination  of  the  colonies 
throughout  the  organ,  E.  Frankel  recommends  that  as 
soon  as  the  organ  is  removed  from  the  body  it  be  wrapped 


TYPHOID  FEVER.  263 

in  cloths  wet  with  a  solution  of  bichlorid  of  mercury  and 
kept  for  three  days  in  a  warm  room,  in  order  that  a  con- 
siderable and  massive  development  of  the  bacilli  may  take 
place. 

Typhoid  fever  is  a  disease  which  is  communicable  to 
animals  with  difficulty.  They  are  not  affected  by  bacilli 
in  fecal  matter  or  in  pure  culture  mixed  with  the  food, 
and  are  not  diseased  by  the  injection  into  them  of  blood 
from  typhoid  patients.  Gaffky  failed  completely  to  pro- 
duce any  symptoms  suggestive  of  typhoid  fever  in  rab- 
bits, guinea-pigs,  white  rats,  mice,  pigeons,  chickens, 
and  calves,  and  found  that  Java  apes  could  feed  daily 
upon  food  polluted  with  typhoid  germs  for  a  considerable 
time,  yet  without  symptoms.  The  introduction  of  pure 
cultures  into  the  abdominal  cavity  of  most  animals  is 
without  effect.  Frankel  and  Simon,  however,  found  that 
when  pure  cultures  are  injected  into  mice,  rabbits,  and 
guinea-pigs  the  animals  die.  Many  observers  attribute 
the  deaths  in  such  cases  to  the  toxin  injected  with  the 
bacilli,  and  consider  it  entirely  independent  of  the  living 
organisms  injected.  In  such  fatal  cases,  however,  the 
bacilli  are  found  in  large  numbers  in  the  blood,  making 
the  condition  resemble  septicemia. 

When  animals  are  treated  in  the  manner  described  in 
the  chapter  upon  Cholera — i.  e.  the  gastric  contents  ren- 
dered alkaline,  a  large  quantity  of  laudanum  injected 
into  the  peritoneal  cavity,  and  the  bacilli  introduced 
through  an  esophageal  catheter — Klemperer,  Levy,  and 
others  found  that  there  was  produced  an  intestinal  con- 
dition which  very  much  resembled  typhoid  as  it  occurs  in 
man.  The  virulence  of  the  bacillus  can  be  very  greatly 
increased  by  rapid  passage  from  guinea-pig  to  guinea-pig. 

In  the  experiments  of  Chantemesse  and  Widal  the 
symptoms  following  the  injection  of  virulent  culture  into 
guinea-pigs  were  briefly  as  follows :  ' '  Very  shortly  after 
the  inoculation  there  is  a  rise  of  temperature,  which 
continues  from  one  to  four  hours,  and  is  succeeded  by  a 
depression  of  the  temperature,  which  continues  to  the 


264  PATHOGENIC  BACTERIA. 

fatal  issue.  Meteorism  and  great  tenderness  of  the  abdo- 
men are  observed.  At  the  autopsy  a  sero-fibrinous  or 
sero-purulent  peritonitis  is  observed — sometimes  hemor- 
rhagic. There  is  also  generally  a  pleurisy,  either  serous 
or  hemorrhagic.  All  the  abdominal  viscera  are  con- 
gested. The  intestine  is  congested — contains  an  abun- 
dant mucous  secretion.  The  Peyer  patches  are  enlarged. 
The  spleen  is  enlarged,  blackish,  and  often  hemorrhagic. 
In  cases  which  are  prolonged  the  liver  is  discolored.  The 
kidneys  are  congested,  the  adrenals  filled  with  blood. 

* '  In  such  cases  the  bacillus  can  be  found  upon  the  in- 
flamed serous  membranes,  in  the  inflammatory  exudates, 
in  the  spleen  in  large  numbers,  in  the  adrenals,  the  liver, 
the  kidneys,  and  sometimes  in  the  lungs.  The  blood  is 
also  infected,  but  to  a  rather  less  degree. 

"In  cases  described  as  chronic,  the  bacillus  disappears 
completely  in  from  five  to  twenty-four  hours,  and  pro- 
duces but  one  lesion,  a  small  abscess  at  the  point  of  inoc- 
ulation. 

4 '  Sanarelli  has  observed  that  if  some  of  the  poisonous 
products  of  the  colon  bacillus  or  the  Proteus  vulgaris  be 
injected  into  the  abdominal  cavity  of  an  animal  recover- 
ing from  a  chronic  case,  it  speedily  succumbs  to  typical 
typhoid  fever." 

The  failure  to  produce  a  satisfactory  combination  of 
symptoms  by  experimental  inoculation  into  animals  is 
one  of  the  impediments  in  the  way  of  the  production 
of  an  antitoxin  for  use  in  human  medicine.  As  long  as 
there  can  be  the  slightest  doubt  thrown  upon  the  speci- 
ficity of  the  bacillus  because  of  the  failure  to  produce 
the  recognized  symptoms  in  animals,  so  long  an  anti- 
toxic substance,  if  produced  at  all,  will  be  rejected  by 
many  in  the  profession.  Animals  can  easily  be  accus- 
tomed to  this  bacillus,  and  when  so  accustomed  seem, 
according  to  Chantemesse  and  Widal,  to  develop  in  their 
blood  an  antitoxic  substance  capable  of  protecting  other 
animals.  Stern  has  also  found  that  in  the  blood  of  re- 
cent human  convalescents  a  substance  exists  which  has  a 


TYPHOID  FEVER.  265 

distinct  protective  effect  upon  guinea-pigs.  His  observa- 
tion is  in  accordance  with  an  earlier  one  in  the  same  line 
by  Chantemesse  and  Widal.  There  is  only  the  foreshad- 
owing of  a  useful  antitoxic  substance  in  the  work  which 
has  already  been  done,  but,  judging  from  the  success 
met  with  in  tetanus  and  diphtheria,  we  can  build  exalted 
hopes  of  future  success. 

Rumpf,  Kraus,  and  Buswell  report  a  number  of  cases 
of  typhoid  which  were  favorably  influenced  by  the  intro- 
duction hypodermically  of  small  quantities  of  sterilized 
cultures  of  Bacillus  pyocyaneus,  and  have  thus  added 
somewhat  to  our  knowledge  of  antagonistic  bacteria  and 
neutralizing  toxins.  These  experiments  are  still  too  new 
to  deserve  prolonged  mention. 

One  of  the  most  important  and  practical  points  for  the 
physician  to  grasp  in  relation  to  the  subject  of  typhoid 
fever  is  the  highly  virulent  character  of  the  discharges 
from  the  bowels.  In  every  case  the  greatest  care  should 
be  taken  for  a  proper  disinfection  of  the  feces,  a  rigid 
attention  to  all  the  details  of  cleanliness  in  the  sick- 
room, and  the  careful  sterilization  of  all  articles  which 
are  soiled  by  the  patient.  If  country  practitioners  were 
as  careful  in  this  particular  as  they  should  be,  the  disease 
would  be  much  less  frequent  in  regions  remote  from  the 
filth  and  squalor  of  the  large  cities  with  their  unmanage- 
able slums,  and  the  distribution  of  the  bacilli  to  villages 
and  towns  by  watercourses  polluted  in  their  infancy 
might  be  checked. 


CHAPTER  VI. 
CHOLERA. 

CHOLERA  is  a  disease  from  which  certain  parts  of  India 
are  never  free.  These  areas,  in  which  it  is  endemic,  are 
the  foci  from  which  the  great  epidemics  of  the  world,  as 
well  as  the  constant  smaller  epidemics  of  India,  probably 
spread.  No  one  knows  when  cholera  was  first  introduced 
into  India,  and  the  probabilities  are  that  it  is  indigenous 
to  that  country,  as  yellow  fever  is  to  Cuba.  Very  early 
mention  of  it  is  made  in  the  letters  of  travellers,  in 
books  and  papers  on  medicine  of  a  century  ago,  and 
in  the  governmental  statistics,  yet  we  find  that  little  is 
said  about  the  disease  except  in  a  general  way,  most 
attention  being  directed  to  the  effect  upon  the  armies, 
native  and  European,  of  India  and  adjacent  countries. 
The  opening  up  of  India  by  Great  Britain  in  the  last 
half  century  has  made  possible  much  accurate  scientific 
observation  of  the  disease  and  the  relation  which  its  epi- 
demics bear  to  the  manners  and  customs  of  the  people. 

The  filthy  habits  of  the  people  of  India,  their  poverty, 
their  crowded  condition,  and  their  religious  customs,  all 
serve  to  aid  in  the  distribution  of  the  disease.  We  are 
told  that  the  city  of  Benares  drains  into  the  Ganges  River 
by  a  most  imperfect  system,  which  distributes  the  greater 
part  of  the  sewage  immediately  below  the  banks  upon 
which  the  city  is  built.  It  is  a  matter  of  religious  ob- 
servance for  every  zealot  who  makes  a  pilgrimage  to  the 
"sacred  city  "  to  take  a  bath  in  and  drink  a  large  quan- 
tity of  this  sacred  but  polluted  water,  and,  as  may  be 
imagined,  the  number  of  pious  Hindoos  who  leave 
Benares  with  comma  bacilli  in  their  intestines  or  upon 
their  clothes  is  great,  for  there  are  few  months  in  the 

266 


CHOLERA.  267 

year  when  there  are  not  at  least  some  cases  of  cholera 
in  the  city. 

The  frequent  pilgrimages  and  great  festivals  of  the 
Hindoos  and  Moslems,  by  bringing  together  an  enormous 
number  of  people  who  crowd  in  close  quarters  where  filth 
and  bad  diet  are  common,  cause  a  rapid  increase  in  the 
number  of  cases  during  these  periods  and  the  dispersion 
of  the  disease  when  the  festivals  break  up.  The  disease 
extends  readily  along  the  regular  lines  of  travel,  visiting 
town  after  town,  until  from  Asia  it  has  frequently  ex- 
tended into  Europe,  and  by  the  steamships  plying  on 
foreign  waters  has  been  several  times  carried  to  our  own 
continent  and  to  the  islands  of  the  seas.  Many  cases  are 
on  record  which  show  conclusively  how  a  single  ship, 
having  a  few  cholera  cases  on  board,  may  be  the  cause 
of  an  outbreak  of  the  disease  in  the  port  at  which  it 
arrives. 

It  seems  strange  to  us  now,  with  the  light  of  present 
information  illuminating  the  pages  of  the  past,  to  observe 
how  the  distinctly  infectious  nature  of  such  a  disease 
could  be  overlooked  in  the  search  for  some  atmospheric 
or  climatic  cause,  some  miasm,  which  was  to  account 
for  it. 

The  discovery  of  the  organism  which  seems  to  be  the 
specific  cause  of  cholera  was  made  by  Koch,  who  was 
appointed  one  of  a  German  cholera-commission  to  study 
the  disease  in  Egypt  and  India  in  1883-84.  Since  his 
discovery,  but  a  decade  ago,  the  works  upon  cholera  and 
the  published  investigations  to  which  the  spirillum  has 
been  subjected  have  produced  an  immense  literature, 
a  large  part  of  which  was  stimulated  by  the  Hamburg 
epidemic  of  two  years  ago. 

The  micro-organism  described  by  Koch,  and  now  gen- 
erally accepted  to  be  the  cause  of  cholera,  is  a  short 
individual  about  half  the  length  of  a  tubercle  bacillus, 
considerably  stouter,  and  distinctly  curved,  so  that  the 
original  name  by  which  it  was  known  was  the  '  *  comma 
bacillus"  (Figs.  77,  78). 


268  PATHOGENIC  BACTERIA. 

A  study  of  the  growth  of  the  organism  and  the  forms 
which  it  assumes  upon  different  culture-media  soon  con- 
vinces us  that  we  have  to  do  with  an  organism  in  no  way 
related  to  the  bacilli.  If  the  conditions  of  nutrition  are 


FIG.  77. — Spirillum  of  Asiatic  cholera,  showing  the  flagella;  x  1000  (Giinther). 

diminished  so  that  the  multiplication  of  the  bacteria  by 
simple  division  does  not  progress  with  the  usual  rapidity, 
we  find  a  distinct  tendency  toward — and  in  some  cases, 
as  upon  potato,  a  luxuriant  development  of — long  spiral 
threads  with  numerous  windings — unmistakable  spirilla. 
Frankel  has  found  that  the  exposure  of  cultures  to  unusu- 
ally high  temperatures,  the  addition  of  small  amounts 
of  alcohol  to  the  culture-media,  etc.,  will  so  vary  the 
growth  of  the  organism  as  to  favor  the  production  of 
spirals  instead  of  commas.  One  of  the  most  common 
of  the  numerous  forms  observed  is  that  in  which  two 
short  curved  individuals  are  so  joined  as  to  produce  an 
S-shaped  curve. 

The  cholera  spirilla  are  exceedingly  active  in  their 
movements,  and  in  hanging-drop  cultures  can  be  seen 
to  swim  about  with  great  rapidity.  Not  only  do  the 
comma-shaped  organisms  move,  but  when  distinct  spirals 
exist,  they,  too,  move  with  the  rapid  rotary  motion  so 
common  among  the  spirilla. 


CHOLERA,  269 

The  presence  of  flagella  upon  the  cholera  spirillum 
can  be  demonstrated  without  difficulty  by  L,6ffler's 
method  (q.  v.).  Bach  spirillum  possesses  a  single  flagel- 
lum  attached  to  one  end. 

Inoculation-forms  of  most  bizarre  appearance  are  very 
common  in  old  cultures  of  the  spirillum,  and  very  often 


FIG.  78. — Spirillum  of  Asiatic  cholera,  from  a  bouillon  culture  three  weeks  old, 
showing  numbers  of  long  spirals;    x  1000  (Frankel  and  Pfeiffer). 

there  can  be  found  in  fresh  cultures  many  individuals 
which  show  by  granular  protoplasm  and  irregular  outline 
that  they  are  partly  degenerated.  Cholera  spirilla  from 
various  sources  seem  to  differ  in  this  particular,  some 
of  the  forms  being  as  pronounced  in  their  involution 
as  the  diphtheria  bacilli. 

In  partially  degenerated  cultures  in  which  long  spirals 
are  numerous  Hiippe  observed,  by  examination  in  the 
" hanging  drop,"  in  the  continuity  of  the  elongate  mem- 
bers, certain  large  spherical  bodies  which  he  described  as 
spores.  These  bodies  were  not  enclosed  in  the  organisms 
like  the  spores  of  anthrax,  but  seemed  to  exemplify  the 
form  of  sporulation  in  which  an  entire  individual  trans- 
forms itself  into  a  spore  (arthrospore).  Koch,  and  indeed 
all  other  observers,  failed  to  find  signs  of  fructification  in 


27°  PATHOGENIC  BACTERIA. 

the  cholera  organism,  and  the  true  nature  of  the  bodies 
described  by  Hiippe  must  be  regarded  as  doubtful. 
Most  bacteriologists  disagree  with  Hiippe  in  believing 
that  arthrospores  exist  at  all,  and  the  fact  (which  will  be 
pointed  out  later  on)  that  there  is  very  little  permanence 
about  cholera  cultures  throws  additional  doubt  upon  the 
accuracy  of  Hiippe' s  conclusion. 

The  cholera  spirillum  stains  well  with  the  ordinary 
aqueous  solutions  of  the  anilin  dyes  ;  fuchsin  seems  par- 
ticularly appropriate.  At  times  the  staining  must  be  con- 
tinued for  from  five  to  ten  minutes  to  secure  homogeneity. 
The  cholera  spirillum  does  not  stain  by  Gram's  method. 
It  may  be  colored  and  examined  while  alive ;  thus  Cornil 
and  Babes,  in  demonstrating  it  in  the  rice-water  dis- 
charges, "spread  out  one  of  the  white  mucous  fragments 
upon  a  glass  slide  and  allow  it  to  dry  partially  ;  a  small 
quantity  of  an  exceedingly  weak  solution  of  methyl  violet 
in  distilled  water  is  then  flowed  over  it,  and  it  is  flattened 
out  by  pressing  down  on  it  a  cover-glass,  over  which  is 
placed  a  fragment  of  filter-paper,  which  absorbs  any 
excess  of  fluid  at  the  margin  of  the  cover-glass.  Comma 
bacilli  so  prepared  and  examined  with  an  oil-immersion 
lens  (x  700-800)  may  then  be  seen :  their  characters  are 
the  more  readily  made  out  because  of  the  slight  stain 
which  they  take  up,  and  because  they  still  retain  their 
power  of  .vigorous  movement,  which  would  be  entirely 
lost  if  the  specimen  were  dried,  stained,  and  mounted  in 
the  ordinary  fashion." 

The  colonies  of  the  spirillum  when  grown  upon  gel- 
atin plates  are  highly  characteristic.  They  appear  in 
the  lower  strata  of  the  gelatin  as  small  white  dots,  grad- 
ually grow  out  to  the  surface,  effect  a. gradual  liquefaction 
of  the  medium,  and  then  appear  to  be  situated  in  little 
pits  with  sloping  sides  (Fig.  79).  This  peculiar  appear- 
ance, which  gives  one  the  suggestion  that  the  plate  is 
full  of  little  holes  or  air-bubbles,  is  due  to  the  evapora- 
tion of  the  liquefied  gelatin. 

One  of  the  best  methods  of  securing  pure  cultures  of 


CHOLERA.  271 

the  cholera  spirillum,  and  also  of  making  a  diagnosis 
of  the  disease  in  a  suspected  case,  is  probably  that  of 
Schottelius.  The  method  is  very  simple  :  A  small  quan- 
tity of  the  fecal  matter  is  mixed  with  bouillon  and  stood 
in  an  incubating  oven  for  twenty-four  hours.  If  the 


FIG.  79. — Spirillum  of  Asiatic  cholera :  colonies  two  days  old  upon  a  gelatin 
plate;    x  35  (Heim). 

cholera  spirilla  are  present,  they  will  grow  most  rapidly 
at  the  surface  of  the  liquid  when  the  supply  of  air  is 
good.  A  pellicle  will  be  formed,  a  drop  from  which, 
diluted  in  melted  gelatin  and  poured  upon  plates,  will 
show  typical  colonies. 

Under  the  microscope  the  principal  characteristics 
can  be  made  out.  The  colony  of  the  cholera  spirillum 
scarcely  resembles  that  of  any  other  organism.  The  little 
colonies  which  have  not  yet  reached  the  surface  of  the 
gelatin  begin  very  soon  to  show  a  pale-yellow  color  and 
to  exhibit  irregularities  of  contour,  so  that  they  are 
almost  never  smooth  and  round.  They  are  coarsely 
granular,  and  have  the  largest  granules  in  the  centre. 
As  the  colony  increases  in  size  the  granules  also  increase 


272  PATHOGENIC  BACTERIA. 

in  size,  and  attain  a  peculiar  transparent  character  which 
is  suggestive  of  powdered  glass.  The  commencement 
of  liquefaction  causes  the  colony  to  be  surrounded  with  a 
transparent  halo.  When  this  occurs  the  colony  begins  to 
sink,  from  the  digestion  and  evaporation  of  the  medium, 
and  also  to  take  on  a  peculiar  rosy  color. 

In  puncture-cultures  in  gelatin  the  growth  is  again  so 
characteristic  that  it  is  quite  diagnostic  (Fig.  80).     The 


FIG.  80. — Spirillum    cholera  Asiatica ;    gelatin   puncture-cultures    aged   forty- 
eight  and  sixty  hours  (Shakespeare). 


growth  takes  place  along  the  entire  puncture,  but  devel- 
ops best  at  the  surface,  where  it  is  in  contact  with  the 
atmosphere.  An  almost  immediate  liquefaction  of  the 
medium  begins,  and,  keeping  pace  with  the  rapidity  of 
the  growth,  is  more  marked  at  the  surface  than  lower 
down.  The  result  of  this  is  the  occurrence  of  a  short, 
rather  wide  funnel  at  the  top  of  the  puncture.  As  the 
growth  continues  evaporation  of  the  medium  takes  place 
slowly,  so  that  the  liquefied  gelatin  is  lower  than  the 
solid  surrounding  portions,  and  appears  to  be  surmounted 
by  an  air-bubble. 


CHOLERA.  273 

The  luxuriant  development  of  the  spirilla  in  gelatin 
produces  considerable  solid  material  to  sediment  and  fill 
up  the  lower  third  or  lower  half  of  the  liquefied  area. 
This  solid  material  consists  of  masses  of  spirilla  which 
have  probably  completed  their  life-cycle  and  become 
inactive.  Under  the  microscope  they  exhibit  the  most 
varied  involution-forms.  The  liquefaction  reaches  the 
sides  of  the  tube  in  from  five  to  seven  days.  Liquefac- 
tion of  the  medium  is  not  complete  for  several  weeks. 
According  to  Frankel,  in  eight  weeks  the  organisms  in 
the  liquefied  culture  all  die,  and  cannot  be  transplanted. 
Kitasato,  however,  has  found  them  living  and  active  on 
agar-agar  after  ten  to  thirty  days,  and  Koch  was  able 
to  demonstrate  their  vitality  after  two  years. 

When  planted  upon  the  surface  of  agar-agar  the  spi- 
rilla produce  a  white,  shining,  translucent  growth  along 
the  entire  line  of  inoculation.  It  is  in  no  way  peculiar. 
The  vitality  of  the  organism  is  retained  much  better  upon 
agar-agar  than  upon  gelatin,  and,  according  to  Frankel, 
the  organism  can  be  transplanted  and  grown  when  nine 
months  old. 

The  growth  upon  blood-serum  likewise  is  without  dis- 
tinct peculiarities,  and  causes  gradual  liquefaction  of  the 
medium. 

Upon  potato  the  spirilla  grow  well,  even  when  the 
reaction  of  the  potato  is  acid.  In  the  incubator  at  a 
temperature  of  37°  C,  a  transparent,  slightly  brownish 
or  yellowish-brown  growth,  somewhat  resembling  the 
growth  of  glanders,  is  produced.  It  contains  large 
numbers  of  long  spirals. 

In  bouillon  and  in  peptone  solution  the  cholera  organ- 
isms grow  well,  especially  upon  the  surface,  where  a 
folded,  wrinkled  mycoderma  is  formed.  Below  the  my- 
coderma  the  culture  fluid  generally  remains  clear.  If 
the  glass  be  shaken  and  the  mycoderma  broken  upr 
fragments  of  it  sink  to  the  bottom. 

In  milk  the  development  is  also  luxuriant,  but  takes 
place  in  such  a  manner  as  not  visibly  to  alter  its  appear- 

18 


274  PATHOGENIC  BACTERIA. 

ance.  The  existence  of  cholera  organisms  in  milk  is, 
however,  rather  short-lived,  for  the  occurrence  of  any 
acidity  at  once  destroys  them. 

WolfFhugel  and  Riedel  have  shown  that  if  the  spirilla 
are  plunged  in  sterilized  water  they  grow  with  great  ra- 
pidity after  a  short  time,  and  can  be  found  alive  after 
months  have  passed.  Frankel  points  out  that  this  ability 
to  grow  and  remain  vital  for  long  periods  in  sterilized 
water  does  not  guarantee  the  same  power  in  unsterilized 
water,  for  in  the  latter  the  simultaneous  growth  of  other 
bacteria  in  a  few  days  serves  to  extinguish  the  cholera 
germs. 

One  of  the  characteristics  of  the  cholera  spirillum  is 
the  metabolic  production  of  indol.  The  detection  of  this 
substance  is  easy  if  the  spirilla  are  grown  in  a  transparent 
colorless  solution.  As  the  cholera  organisms  also  produce 
nitrites,  all  that  is  necessary  is  to  add  a  drop  or  two  of 
chemically  pure  sulphuric  acid  to  the  culture-medium 
for  the  production  of  the  well-known  reddish  color. 

Several  toxic  products  of  the  metabolism  of  the  spirilla 
have  been  isolated.  Brieger  and  Frankel  have  isolated 
a  toxalbumin  ;  Villiers,  a  toxic  alkaloid  fatal  to  guinea- 
pigs  ;  and  Gamaleia,  two  substances  about  equally  toxic. 

The  cholera  spirilla  can  be  found  with  great  constancy 
in  the  intestinal  evacuations  of  all  cholera  cases,  and  can 
often  be  found  in  the  drinking-water,  milk,  and  upon 
vegetables,  etc.  in  cholera-infected  districts.  There  can 
be  little  doubt  that  they  find  their  way  into  the  body 
through  the  food  and  drink.  Many  cases  are  reported 
in  the  literature  upon  cholera  that  show  how  the  disease- 
germs  enter  the  drinking-water,  and  are  thus  distributed  ; 
how  they  are  sometimes  thoughtlessly  sprinkled  over  veg- 
etables, offered  for  sale  in  the  streets,  with  water  from 
polluted  gutters ;  how  they  enter  milk  with  water  used 
to  dilute  it ;  how  they  are  carried  about  in  clothing  and 
upon  foodstuffs ;  how  they  can  be  brought  to  articles  of 
food  upon  the  table  by  flies  which  have  preyed  upon 
cholera  excrement ;  and  how  many  other  interesting  in- 


CHOLERA.  275 

factions  are  made  possible.  The  literature  upon  these 
subjects  is  so  vast  that  in  a  sketch  of  this  kind  it  is 
scarcely  possible  to  comprise  even  the  most  instructive 
examples.  One  physician  is  reported  to  have  been  in- 
fected with  cholera  while  experimenting  with  the  spirilla 
in  Koch's  laboratory. 

The  evidence  of  the  specificity  of  the  cholera  spirillum 
when  collected  shows  that  it  is  present  in  the  choleraic 
dejections  with  great  regularity,  and  that  it  is  as  con- 
stantly absent  from  the  dejecta  of  healthy  individuals 
and  those  suffering  from  other  diseases ;  but  these  facts 
do  not  admit  of  satisfactory  proof  by  experimentation 
upon  animals.  Animals  are  never  affected  by  any  dis- 
ease similar  to  cholera  during  the  epidemics,  nor  do  foods 
mixed  with  cholera  discharges  or  with  pure  cultures  of 
the  cholera  spirillum  affect  them.  This  being  true,  we 
are  prepared  to  receive  the  further  information  that  sub- 
cutaneous injections  of  the  spirilla  are  often  without 
serious  consequences,  though  cultures  differ  very  much 
in  this  respect,  some  always  causing  a  fatal  septicemia  in 
guinea-pigs,  others  being  as  constantly  harmless. 

Intraperitoneal  injection  of  the  virulent  cultures  pro- 
duces a  fatal  peritonitis  in  guinea-pigs. 

One  reason  that  animals  and  certain  men  are  immune 
to  the  disease  seems  to  be  found  in  the  distinct  acidity 
of  the  normal  gastric  juice,  and  the  destruction  of  the  spi- 
rilla by  it.  Supposing  that  this  might  be  the  case,  Nicati 
and  Rietsch,  Von  Brmengen  and  Koch,  have  suggested 
methods  by  which  the  micro-organisms  can  be  introduced 
directly  into  the  intestine.  The  first-named  investigators 
ligated  the  common  bile-duct  of  guinea-pigs,  and  then  in- 
jected the  spirilla  into  the  duodenum  with  a  hypodermic 
needle.  The  result  was  that  the  animals  usually  died,  some- 
times with  choleraic  symptoms  ;  but  the  excessively  grave 
nature  of  the  operation  upon  such  a  small  and  delicately 
constituted  animal  as  a  guinea-pig  greatly  lessens  the  value 
of  the  experiment.  Koch's  method  is  much  more  satisfac- 
tory. By  injecting  laudanum  into  the  abdominal  cavity 


276  PATHOGENIC  BACTERIA. 

of  guinea-pigs  the  peristaltic  movements  are  checked. 
The  amount  given  for  the  purpose  is  very  large,  about 
i  gram  for  each  200  grams  of  body-weight.  It  generally 
narcotizes  the  animals  for  a  short  time,  but  they  recover 
without  injury.  After  administering  the  opium  the  con- 
tents of  the  stomach  are  neutralized  by  introducing 
through  a  pharyngeal  catheter  5  c.cm.  of  a  5  per  cent, 
aqueous  solution  of  sodium  carbonate.  With  the  gastric 
contents  thus  alkalinized  and  the  peristalsis  paralyzed  a 
bouillon  culture  of  the  spirilla  is  introduced.  The  ani- 
mal recovers  from  the  manipulation,  but  shows  an  indis- 
position to  eat,  is  soon  observed  to  be  weak  in  the  pos- 
terior extremities,  subsequently  is  paralyzed,  and  dies 
within  forty-eight  hours.  The  autopsy  shows  the  intes- 
tine congested  and  filled  with  a  watery  fluid  rich  in  spi- 
rilla— an  appearance  which  Frankel  declares  to  be  exactly 
that  of  cholera.  In  man,  as  well  as  in  these  artificially 
injected  animals,  the  spirilla  are  never  found  in  the  blood 
or  the  tissues,  but  only  in  the  intestine,  where  they  fre- 
quently enter  between  the  'basement  membrane  and  the 
epithelial  cells,  and  aid  in  the  detachment  of  the  latter. 

Guinea-pigs  are  also  susceptible  to  intraperitoneal  in- 
jections of  the  spirillum,  and  speedily  succumb.  The 
symptoms  are — rapid  fall  of  temperature,  tenderness  over 
the  abdomen,  and '  collapse.  The  autopsy  shows  an 
abundant  fluid  exudate  containing  the  micro-organism, 
and  injection  and  redness  of  the  peritoneum  and  viscera. 

Although  in  reading  upon  cholera  at  the  present  time 
we  find  very  little  skepticism  in  relation  to  Koch's 
"  comma  bacillus,"  we  do  find  occasional  doubters  who 
believe  with  Von  Pettenkoffer  that  the  disease  is  mias- 
matic. PettenkofFer's  theory  is  that  the  disease  has 
much  to  do  with  the  ground-water  and  its  drying  zone. 
He  regards  as  the  principal  cause  of  the  disease  the  de- 
velopment of  germs  in  the  subsoil  moisture  during  the 
warm  months,  and  their  impregnation  of  the  atmosphere 
as  a  miasm  to  be  inhaled,  instead  of  ingested  with  food 
and  drink.  This  idea  of  PettenkofFer's,  combined  with 


CHOLERA.  277 

his  other  idea  that  individual  predisposition  must  pre- 
cede the  inception  of  the  disease,  is  scarcely  compatible 
with  what  has  gone  before,  and  cannot  possibly  be  made 
to  explain  the  march  of  the  disease  from  place  to  place 
with  caravans,  or  its  distribution  over  extended  areas 
when  fairs  and  religious  gatherings  among  the  Hindoos 
break  up,  the  people  from  an  infected  centre  carrying 
cholera  with  them  to  their  homes. 

While  it  is  an  organism  that  multiplies  with  great 
rapidity  under  proper  conditions,  the  cholera  spirillum 
is  not  possessed  of  much  resisting  power.  Sternberg 
found  that  it  was  killed  by  exposure  to  a  temperature 
of  52°  C.  for  four  minutes.  Kitasato,  however,  found 
that  ten  or  fifteen  minutes'  exposure  to  a  temperature 
of  55°  C.  was  not  always  fatal.  In  the  moist  con- 
dition the  organism  may  retain  its  vitality,  for  months, 
but  it  is  very  quickly  destroyed  by  desiccation,  as  was 
found  by  Koch,  who  observed  that  when  dried  in  a  thin 
film  its  power  to  grow  was  destroyed  in  a  few  hours. 
Kitasato  found  that  upon  silk  threads  the  vitality  might 
be  retained  longer.  Abel  and  Claussen  have  shown  that 
it  does  not  live  longer  than  twenty  to  thirty  days  in  fecal 
matter,  and  often  disappears  in  one  to  three  days.  The 
organism  is  very  susceptible  to  the  influence  of  carbolic 
acid,  bichlorid  of  mercury,  and  other  germicides. 

This  low  vital  resistance  of  the  microbe  is  very  fortu- 
nate, for  it  enables  us  to  establish  safeguards  for  the  pre- 
vention of  the  spread  of  the  disease.  Excreta,  soiled 
clothing,  etc.  are  readily  rendered  harmless  by  the  proper 
use  of  disinfectants.  Water  and  foods  are  rendered  in- 
nocuous by  boiling  or  cooking.  Vessels  may  be  disin- 
fected by  thorough  washings  with  jets  of  boiling  water 
thrown  upon  them  through  hose.  Baggage  can  be  steril- 
ized by  superheated  steam. 

It  often  becomes  a  matter  of  importance  to  detect  the 
presence  of  cholera  in  drinking-water,  and,  as  the  dilu- 
tion in  which  the  bacteria  exist  in  such  a  liquid  may  be 
very  great,  much  difficulty  is  experienced  in  finding  them 


278  PATHOGENIC  BACTERIA. 

by  ordinary  methods.  One  of  the  most  expeditious  meth- 
ods that  have  been  recommended  is  that  of  Loffler,  who 
adds  200  c.cm.  of  the  water  to  be  examined  to  10  c.cm. 
of  bouillon,  allows  the  mixture  to  stand  in  an  incubator 
for  twelve  to  twenty-four  hours,  and  then  makes  plate- 
cultures  from  the  superficial  layer  of  the  liquid,  where, 
if  present,  the  development  of  the  spirilla  will  be  most 
rapid  because  of  the  presence  of  air.  A  similar  method 
can  be  used  to  detect  the  spirilla  when  their  presence  is 
suspected  in  feces. 

Gruber  and  Wiener,  Haff kine,  Pawlowsky,  and  Pfeiffer 
have  all  succeeded  in  immunizing  animals  against  the 
toxic  substances  removed  from  cholera  cultures  or  against 
living  cultures  properly  injected.  There  seems,  accord- 
ing to  the  researches  of  Pfeiffer,  to  be  no  doubt  that  in 
the  blood  of  the  protected  animals  a  protective  substance 
is  present.  In  the  peritoneal  infection  of  guinea-pigs 
the  spirilla  grow  vigorously  in  the  peritoneal  cavity,  and 
can  be  found  in  immense  numbers  after  twelve  to  twenty- 
four  hours.  If,  however,  together  with  the  culture  used 
for  inoculation,  a  few  drops  of  the  protective  serum  be  in- 
troduced, Pfeiffer  found  that  instead  of  multiplying  the 
organisms  underwent  a  peculiar  granular  degeneration 
and  disappeared,  the  unprotected  animal  dying,  the  pro- 
tected animal  remaining  well. 

Of  the  numerous  attempts  which  have  from  time  to 
time  been  made,  and  are  still  being  made,  to  produce 
immunity  against  cholera  in  man  or  to  cure  cholera 
when  once  established  in  the  human  organism,  nothing 
very  favorable  can  at  the  present  time  be  said.  Experi- 
ments in  this  field  are  not  new  :  we  find  Dr.  Ferran  ad- 
ministering hypodermic  injections  of  pure  virulent  cul- 
tures of  the  cholera  spirillum  in  Spain  as  early  as  1885, 
in  the  hope  of  bringing  about  immunity.  The  more  mod- 
ern work  of  Haffkine  seems  to  be  followed  by  a  distinct 
diminution  of  mortality  in  protected  individuals.  Ac- 
cording to  the  work  of  this  investigator,  two  vaccines  are 
used,  one  of  which,  being  mild,  prepares  the  animal  (or 


CHOLERA.  279 

man)  for  a  powerful  vaccine,  which,  were  it  not  preceded 
by  the  weaker  form,  would  bring  about  extensive  tissue- 
necrosis  and  perhaps  death.  Protection  certainly  seems 
to  follow  the  operation  of  these  vaccines. 

Haffkine's  studies  embrace  more  than  40,000  inocula- 
tions performed  in  India.  From  his  latest  paper  (Dec. , 
1895)  the  following  extract  will  show  the  results : 

ui.  In  all  those  instances  where  cholera  has  made  a 
large  number  of  victims,  that  is  to  say,  where  it  has 
spread  sufficiently  to  make  it  probable  that  the  whole 
population,  inoculated  and  uninoculated,  were  equally 
exposed  to  the  infection, — in  all  these  places  the  results 
appeared  favorable  to  inoculation. 

U2.  The  treatment  applied  after  an  epidemic  actually 
breaks  out  tends  to  reduce  the  mortality  even  during  the 
time  which  is  claimed  for  producing  the  full  effect  of  the 
operation.  In  the  Goya  Garl,  where  weak  doses  of  a 
relatively  weak  vaccine  had  been  applied,  this  reduction 
was  to  half  the  number  of  deaths  ;  in  the  coolies  of  the 
Assam-Burmah  survey-party,  where,  as  far  as  I  can  gather 
from  my  preliminary  information,  strong  doses  have  been 
applied,  the  number  of  deaths  was  reduced  to  one-seventh. 
This  fact  would  justify  the  application  of  the  method  in- 
dependently of  the  question  as  to  the  exact  length  of  time 
during  which  the  effect  of  this  vaccination  lasts. 

"3.  In  L,ucknow,  where  the  experiment  was  made  on 
small  doses  of  weak  vaccines,  a  difference  in  cases  and 
deaths  was  still  noticeable  in  favor  of  the  inoculated 
fourteen  to  fifteen  months  after  vaccination  in  an  epidemic 
of  exceptional  virulence.  This  makes  it  probable  that  a 
protective  effect  could  be  obtained  even  for  long  periods 
of  time  if  larger  doses  of  a  stronger  vaccine  were  used. 

"  4.  The  best  results  seem  to  be  obtained  from  applica- 
tion of  middle  doses  of  both  anticholera  vaccines,  the 
second  one  being  kept  at  the  highest  possible  degree  of 
virulence  obtainable. 

4  *  5.  The  most  prolonged  observations  on  the  effect  of 
middle  doses  were  made  in  Calcutta,  where  the  mortality 


280  PATHOGENIC  BACTERIA. 

from  the  eleventh  up  to  the  four  hundred  and  fifty-ninth 
day  after  vaccination  was,  among  the  inoculated,  17.24 
times  smaller,  and  the  number  of  cases  19.27  times 
smaller  than  among  the  not  inoculated." 

Pawlowsky  and  others  have  found  that  the  dog  is  sus- 
ceptible to  cholera,  and  have  utilized  the  observation  to 
prepare  an  antitoxic  serum  in  considerable  quantities. 
The  dogs  were  first  immunized  with  attenuated  cultures, 
then  with  more  and  more  virulent  cultures,  until  a  serum 
was  obtained  whose  value  was  estimated  at  i  :  130,000 
upon  experimental  animals. 

Freymuth  and  others  have  endeavored  to  secure  favor- 
able results  from  the  injection  of  blood-serum  from  con- 
valescent patients  into  the  diseased.  One  recovery  out 
of  three  cases  treated  is  recorded — not  a  very  glittering 
result. 

In  all  these  preliminaries  the  foreshadowing  of  a  future 
therapeusis  must  be  evident,  but  as  yet  nothing  really 
satisfactory  has  been  achieved. 


CHAPTER    VII. 
SPIRILLA  RESEMBLING  THE  CHOLERA  SPIRILLUM. 

The  Finkler  and  Prior  Spirillum. — Somewhat  similar 
to  the  spirillum  of  cholera,  and  in  some  respects  closely 
related  to  it,  is  the  spirillum  obtained  from  the  feces  of 
a  case  of  cholera  nostras  by  Finkler  and  Prior  in  1884. 
It  is  a  rather  shorter,  stouter  organism,  with  a  more  pro- 
nounced curve,  than  the  cholera  spirillum,  and  rarely 
forms  the  long  spirals  which  characterize  the  latter. 
The  central  portion  is  also  somewhat  thinner  than  the 
ends,  which  are  a  little  pointed  and  give  the  organism 
a  less  uniform  appearance  than  that  of  cholera  (Fig.  81). 


— €  t  *• .  .   :w%  ?<*.  «wi> 


FIG.  8l. — Spirillum  of  Finkler  and  Prior,  from  an  agar-agar  culture;    x  1000 
(Itzerott  and  Niemann). 

Involution-forms  are  very  common  in  cultures,  and  occur 
as  spheres,  spindles,  clubs,  etc.  Like  the  cholera  spiril- 
lum, each  organism  is  provided  with  a  single  flagellum 

-     281 


282  PATHOGENIC  BACTERIA. 

situated  at  its  end,  and  is  actively  motile.  Although  at 
first  thought  to  be  a  variety  of  the  cholera  germ,  marked 
differences  of  growth  were  soon  observed,  and  showed 
the  organism  to  be  a  separate  species. 

The  growth  upon  gelatin  plates  is  quite  rapid,  and  leads 
to  such  extensive  liquefaction  that  four  or  five  dilutions 
must  frequently  be  made  before  the  growth  of  a  single 
colony  can  be  observed.  To  the  naked  eye  the  colonies 
appear  as  small  white  points  in  the  depths  of  the  gelatin 
(Fig.  82).  They,  however,  rapidly  reach  the  surface^ 


FIG.  82. — Spirillum  of  Finkler  and  Prior:   colony  twenty-four  hours   old,  as 
seen  upon  a  gelatin  plate;    x   100  (Frankel  and  Pfeiffer). 

begin  liquefaction  of  the  gelatin,  and  by  the  second 
day  appear  about  the  size  of  lentils,  and  are  situated  in 
little  depressions.  Under  the  microscope  they  are  of  a 
yellowish-brown  color,  are  finely  granular,  and  are  sur- 
rounded by  a  zone  of  sharply  circumscribed  liquefied 
gelatin.  Careful  examination  with  a  high  power  of  the 
microscope  shows  a  rapid  movement  of  the  granules  of 
the  colony. 

In  gelatin  punctures  the  growth  takes  place  rapidly 
along  the  whole  puncture,  forming  a  stocking-shaped 
liquefaction  filled  with  cloudy  fluid  which  does  not  pre- 


SPIRILLA  RESEMBLING  CHOLERA.  283 

cipitate  rapidly  ;  a  rather  smeary,  whitish  mycoderma  is 
generally  formed  upon  the  surface.  The  much  more  ex- 
tensive and  more  rapid  liquefaction  of  the  medium,  the 
wider  top  to  the  funnel-shaped  liquefaction  at  the  surface, 


FIG.   83. — Spirillum   of  Finkler  and  Prior :   gelatin  puncture-cultures   aged 
forty-eight  and  sixty  hours  (Shakespeare). 

the  absence  of  the  air-bubble,  and  the  clouded  nature  of 
the  liquefied  material,  all  serve  to  differentiate  it  from  the 
cholera  spirillum. 

Upon  agar-agar  the  growth  is  also  very  rapid,  and  in 
a  short  time  the  whole  surface  of  the  culture-medium  is 
covered  with  a  moist,  thick,  slimy  coating,  which  may 
have  a  slightly  yellowish  tinge. 

The  cultures  upon  potato  are  also  very  different  from 
those  of  cholera,  for  instead  of  a  temperature  of  37°  C. 
being  required  for  a  rapid  development,  the  Finkler  and 
Prior  spirilla  grow  rapidly  at  the  room-temperature,  and 
produce  a  grayish-yellow,  slimy,  shining  layer,  which 
may  cover  the  whole  of  the  culture-medium. 

Blood-serum  is  rapidly  liquefied  by  the  growth  of  the 
organism. 


284  PATHOGENIC  BACTERIA, 

Buchner  has  shown  that  in  media  containing  some 
glucose  an  acid  reaction  is  produced. 

The  spirillum  does  not  grow  well,  if  at  all,  in  milk, 
and  speedily  dies  in  water. 

The  organism  does  not  produce  indol. 

The  spirillum  can  be  stained  well  by  the  ordinary 
dyes,  and  seems,  like  the  cholera  spirillum,  to  have  a 
special  affinity  for  the  aqueous  solution  of  fnchsin. 

In  connection  with  this  bacillus  the  question  of  patho- 
genesis  is  a  very  important  one.  At  first  it  was  sus- 
pected that  it  was,  if  not  the  spirillum  of  cholera  itself, 
a  very  closely  allied  organism.  Later  it  was  regarded 
as  the  cause  of  cholera  nostras.  At  present  its  exact 
pathological  significance  is  a  question.  It  was  in  one 
case  secured  by  Knisl  from  the  feces  of  a  suicide,  and 
has  been  found  in  carious  teeth  by  Miiller. 

When  injected  into  the  stomach  of  guinea-pigs  treated 
according  the  method  of  Koch,  about  30  per  cent,  of  the 
animals  die,  but  the  intestinal  lesions  produced  are  not 
the  same  as  those  produced  by  the  cholera  spirillum. 
The  intestines  in  such  cases  are  pale  and  filled  with 
watery  material  having  a  strong  putrefactive  odor.  This 
fluid  teems  with  the  spirilla. 

It  seems  very  unlikely,  from  the  collected  evidence, 
that  the  Finkler  and  Prior  spirillum  is  associated  with 
pathogenesis  in  the  human  species.  As  Frankel  points 
out,  it  is  probably  a  frequent  and  harmless  inhabitant  of 
the  human  intestine. 

The  Spirillum  of  Denecke. — Another  organism  with 
a  distinct  resemblance  to  the  cholera  spirillum  is  one 
described  by  Denecke  as  occurring  in  old  cheese  (Fig. 
84).  Its  form  is  much  the  same  as  that  of  the  spirillum 
of  cholera,  the  shorter  individuals  being  of  equal  diameter 
throughout.  The  spirals  which  are  produced  are  longer 
than  those  of  the  Finkler  and  Prior  spirillum,  and  are 
more  tightly  coiled  than  those  of  the  cholera  spirillum. 

L,ike  its  related  species,  this  micro-organism  is  actively 
motile.  It  grows  at  the  room-temperature,  as  well  as  at 


SPIRILLA  RESEMBLING  CHOLERA.  285 

37°  C.,  in  this  respect,  as  in  its  reaction  to  stains,  much 
resembling  the  other  two. 

Upon  gelatin  plates  the  growth  of  the  colonies  is  much 
more  rapid  than  that  of  the  cholera  spirillum,  but  slower 
than  that  of  the  Finkler  and  Prior  spirillum.  The  col- 


FIG.   84. — Spirillum  Denecke,  from  an  agar-agar  culture;    x  1000  (Itzerott 
and  Niemann). 

onies  appear  as  small  whitish,  round  points,  which  soon 
reach  the  surface  of  the  gelatin  and  commence  liquefac- 
tion. By  the  second  day  they  are  about  the  size  of  a 
pin's  head,  have  a  yellow  color,  and  occupy  the  bottom 
of  a  conical  depression.  The  appearance  is  much  like 
that  of  a  plate  of  cholera  spirilla. 

The  microscope  shows  the  colonies  to  be  of  irregular 
shape  and  coarsely  granular.  The  color  is  yellow,  and  is 
pale  at  the  edges,  gradually  becoming  intense  toward  the 
centre.  The  colonies  are  surrounded  at  first  by  distinct 
lines  of  circumscription,  later  by  clear  zones,  which,  ac- 
cording to  the  illumination,  are  pale  or  dark.  From  this 
description  it  will  be  seen  that  the  colonies  differ  from 
those  of  cholera  in  the  prompt  liquefaction  of  the  gelatin, 
their  rapid  growth,  yellow  color,  irregular  form,  and  dis- 
tinct lines  of  circumscription. 

In  gelatin  punctures  the  growth  takes  place  all  along 


286  PATHOGENIC  BACTERIA. 

the  track  of  the  wire,  and  forms  a  cloudy  liquid  which 
precipitates  at  the  apex  in  the  form  of  a  coiled  mass. 
Upon  the  surface  a  delicate  imperfect  yellowish  myco- 


FlG.  85. — Spirillum  Denecke  :    gelatin   puncture-cultures  aged  forty-eight  and 
sixty  hours  (Shakespeare). 

derma  forms.  Liquefaction  of  the  entire  gelatin  gen- 
erally requires  about  two  weeks. 

Upon  agar-agar  this  spirillum  grows  as  a  thin  yellow- 
ish layer  which  does  not  seem  inclined  to  spread  widely. 

The'  culture  upon  potato  is  luxuriant  if  grown  in  the 
incubating  oven.  It  appears  as  a  distinct  yellowish  moist 
film,  and  when  examined  microscopically  is  seen  to  con- 
tain long  beautiful  spirals. 

The  organism  sometimes  produces  indol,  but  is  irreg- 
ular in  its  action  in  this  respect. 

The  spirillum  of  Denecke  is  mentioned  only  because 
of  its  morphological  relation  to  the  cholera  spirillum, 
not  because  of  any  pathogenesis  which  it  possesses.  It 
probably  is  not  associated  with  any  human  disease.  Ex- 
periments, however,  have  shown  that  when  the  spirilla 
are  introduced  into  the  intestines  of  guinea-pigs  whose 
gastric  contents  are  alkalinized  and  whose  peristalsis  is 


SPIRILLA  RESEMBLING  CHOLERA.  287 

paralyzed  with  opium,  about  20  per  cent,  of  the  animals 
die  from  intestinal  disease. 

The  Spirillum  of  Gamal£ia  (Spirillum  Metchnikoff). 
— Very  closely  related  to  the  cholera  spirillum  in  its 
morphology  and  vegetation  and  possibly,  as  has  been 
suggested,  a  descendant  of  the  same  original  stock,  is  the 
spirillum  which  Gamaleia  cultivated  from  the  intestines 
of  chickens  affected  with  a  disease  similar  to  chicken- 
cholera.  This  spirillum  is  a  curved  organism,  a  trifle 
shorter  and  thicker  than  the  cholera  spirillum,  a  little 
more  curved,  and  with  similar  rounded  ends  (Fig.  86). 


FIG.  86. — Spirillum  Metchnikoff,  from  an  agar-agar  culture;    x  1000  (Itzerott 
and  Niemann). 

It  forms  long  spirals  in  appropriate  media,  and  is  actively 
motile.  Each  spirillum  is  provided  with  a  terminal  flagel- 
lum.  No  spores  have  been  positively  demonstrated. 

The  organism,  like  the  cholera  vibrio,  is  very  suscep- 
tible to  the  influence  of  acids,  high  temperatures,  and 
drying,  so  that  spores  are  probably  not  formed.  It  grows 
well  both  at  the  temperature  of  the  room  and  at  that  of 
incubation. 

The  bacterium  stains  easily,  the  ends  more  deeply  than 
the  centre.  It  is  not  stained  by  Gram's  method. 

Upon  gelatin   plates  a  remarkable   similarity  to  the 


288  PA  THOGENIC  BACTERIA, 

colonies  of  the  cholera  spirillum  is  developed,  yet  there 
is  a  difference,  and  Pfeiffer  points  out  that  "it  is  com- 
paratively easy  to  differentiate  between  a  plate  of  pure 
cholera  spirillum  and  a  plate  of  pure  Spirillum  Metch- 
nikoff,  yet  it  is  almost  impossible  to  pick  out  a  few 
colonies  of  the  latter  if  mixed  upon  a  plate  with  the 
former. ' ' 

Frankel  regards  this  bacterium  as  a  kind  of  interme- 
diate species  between  the  cholera  and  the  Finkler-Prior 
spirilla. 

The  colonies  upon  gelatin  plates  appear  in  about  twelve 
hours  as  small  whitish  points,  and  rapidly  develop,  so  that 
by  the  end  of  the  third  day  large  saucer-shaped  areas  of 
liquefaction  resembling  colonies  of  the  Finkler-Prior 
spirilla  occur.  The  liquefaction  of  the  gelatin  is  quite 
rapid,  the  resulting  fluid  being  turbid.  Generally  there 
will  be  upon  a  plate  of  Vibrio  Metchnikoff  some  colo- 
nies which  closely  resemble  cholera  by  occupying  small 
conical  depressions  in  the  gelatin.  Under  a  high  power 


FIG.  87. — Spirillum  Metchnikoff;  puncture-culture  in  gelatin  forty-eight  hours 
old  (Frankel  and  Pfeiffer). 

of  the  microscope  the  contents  of  the  colonies,  which  ap- 
pear to  be  of  a  brownish  color,  are  observed  to  be  in  rapid 


SPIRILLA  RESEMBLING  CHOLERA.  289 

motion.  The  edges  of  the  bacterial  mass  are  fringed  with 
radiating  organisms  (Fig.  87). 

In  gelatin  tubes  the  culture  is  very  much  like  that  of 
cholera,  but  develops  more  slowly. 

Upon  the  surface  of  agar-agar  a  yellowish-brown 
growth  develops  along  the  whole  line  of  inoculation. 

On  potato  at  the  room-temperature  no  growth  occurs, 
but  at  the  temperature  of  the  incubator  a  luxuriant 
yellowish-brown  growth  takes  place.  Sometimes  the 
color  is  quite  dark,  and  chocolate-colored  potato  cultures 
are  not  uncommon. 

In  bouillon  the  growth  which  occurs  at  the  tempera- 
ture of  the  incubator  is  quite  characteristic,  and  very 
different  from  that  of  the  cholera  spirillum.  The  entire 
medium  becomes  clouded,  of  a  grayish-white  color,  and 
opaque.  A  folded  and  wrinkled  mycoderma  forms  upon 
the  surface. 

The  addition  of  sulphuric  acid  to  a  culture  grown  in  a 
medium  rich  in  peptone  produces  the  same  rose  color  ob- 
served in  cholera  cultivations. 

The  organism  is  pathogenic  for  animals,  but  not  for 
man.  Pfeiffer  has  shown  that  chickens,  pigeons,  and 
guinea-pigs  are  highly  susceptible  animals.  The  birds 
when  inoculated  under  the  skin  generally  die — pigeons 
always.  When  guinea-pigs  are  treated  according  to 
the  method  of  Koch  for  the  inoculation  of  cholera,  the 
temperature  of  the  animal  rises  for  a  short  time,  then 
abruptly  falls  to  33°  C.  or  less.  Death  follows  in  twenty 
to  twenty-four  hours.  A  distinct  inflammation  of  the 
intestine,  with  exudate  and  numerous  spirilla,  may  be 
found.  The  spirilla  can  also  be  found  in  the  heart's 
blood  and  in  the  organs  of  such  guinea-pigs.  When  the 
bacilli  are  introduced  by  subcutaneous  inoculation,  the 
autopsy  shows  a  bloody  edema  and  a  superficial  necrosis 
of  the  tissues. 

In  the  blood  and  all  the  organs  of  pigeons  and  young 
chickens  the  organisms  can  be  found  in  such  large  num- 
bers that  Pfeiffer  has  suggested  the  term  u  vibrionensep- 

19 


290  PA  THOGENIC  BA  CTERIA. 

ticaemie ' '  for  the  condition.  In  the  intestines  very  few 
alterations  are  noticeable,  and  very  few  spirilla  can  be 
found. 

Garnaleia  has  shown  that  pigeons  and  guinea-pigs  can 
be  made  immune  by  inoculating  them  with  cultures  ster- 
ilized for  a  time  at  a  temperature  of  100°  C.  Mice  and 
rabbits  are  immune  except  to  very  large  doses. 

Spirillum    Berolinensis.— This   organism    (Fig.    88), 


FIG.  88. — Spirillum  Berolinensis,  from  an  agar-agar  culture ;  x   1000  (Itzerott 

and  Niemann). 

which  was  discovered  by  Neisser  in  the  summer  of  1893, 
is  of  great  interest  in  comparison  with  the  spirillum  of 
cholera  and  its  related  forms.  Its  morphology  is  in  every 
particular  exactly  like  that  of  the  cholera  spirillum,  but 
its  growth  is  a  little  more  rapid.  It  grows  upon  the 
same  culture-media  and  at  the  same  temperature.  The 
colonies  are,  however,  quite  different. 

Upon  the  second  day,  when  grown  upon  gelatin 
plates,  the  colonies  of  the  Spirillum  Berolinensis  appear 
finely  granular  and  paler  than  those  of  cholera.  The 
borders  are  generally  smooth  and  circular.  As  it  be- 
comes older  the  colony  takes  on  a  slightly  brownish 
color,  and  may  be  nodulated  or  radiately  lobulated.  The 
gelatin  is  very  slowly  liquefied. 


SPIRILLA   RESEMBLING  CHOLERA.  291 

In  puncture-cultures  the  development  takes  place  along 
the  entire  puncture,  and  causes  a  gradual  liquefaction  of 
the  gelatin. 

Upon  agar-agar  the  growth  is  generally  similar  to  that 
of  the  cholera  spirillum,  but  at  times  is  copious,  dry, 
and'  ragged,  and  suggests  leather  by  its  appearance. 

When  introduced  intraperitoneally  into  guinea-pigs 
the  animals  die  in  from  one  to  two  days. 

The  indol  reaction  is  exactly  like  that  given  by  cul- 
tures of  the  cholera  spirillum.  The  spirillum  does  not 
stain  by  Gram's  method. 

Spirillum  Dunbar. — This  organism  (Fig.  89)  was  de- 


FIG.  89. — Spirillum  Dunbar,  from  agar-agar;  x   1000  (Itzerott  and  Niemann). 

scribed  in  1893  by  Dunbar  and  Oergel,  who  secured  it 
from  the  water  of  the  Elbe  River.  It  much  resembles 
the  cholera  spirillum,  but  it  never  exhibits  sigmoid  forms. 
It  stains  poorly,  the  ends  taking  the  color  much  better 
than  the  central  portion. 

Gelatin  is  liquefied  by  the  growth  of  this  organism 
more  quickly  than  by  the  cholera  spirillum.  The  colo- 
nies upon  gelatin  and  the  puncture-cultures  in  gelatin 
are  identical  with  those  of  the  cholera  spirillum. 

On  agar-agar  a  luxuriant  whitish-yellow  layer  is  pro- 
duced. 


292  PA  THOGENIC  BA  CTERIA. 

In  bouillon  and  peptone  solution  the  addition  of  dilute 
sulphuric  acid  produces  the  red  color  of  nitro-indol. 

It  is  said  that  cultures  grown  at  a  temperature  of  22°  C. 
phosphoresce  in  the  dark. 

The  spirillum  seems  to  be  pathogenic  for  guinea-pigs 
when  introduced  into  the  stomach  according  to  Koch's 
method  for  cholera. 

Spirillum  Danubicus. — This  organism  (Fig.  90)  also 


FIG.  90. — Spirillum  Danubicus,  from  an  agar-agar  culture;  x  1000  (Itzerott  and 

Niemann). 

much  resembles  cholera.  It  was  first  isolated  by  Heider 
in  1892.  In  appearance  it  is  rather  delicate  and  decidedly 
curved.  It  is  often  united  in  sigmoid  and  semicircular 
forms,  and  exhibits  long  spirals  in  old  cultures.  It  is 
actively  motile,  each  organism  presenting  a  terminal 
flagellum. 

The  growth  upon  gelatin  plates  is  rapid.  Small  light- 
gray  colonies,  resembling  those  of  cholera,  but  exhibit- 
ing a  dentate  margin,  are  observed.  The  growth  in 
gelatin  punctures  also  much  resembles  cholera,  and  the 
agar-agar  growth  can  scarcely  be  distinguished  from  it. 

The  potato  growth  has  a  distinct  yellowish-brown 
color. 

Milk  is  coagulated  in  three  or  four  days. 


SPIRILLA  RESEMBLING  CHOLERA.  293 

This  spirillum  does  not  produce  indol. 

Heider  found  the  spirillum  pathogenic  for  guinea-pigs. 

Spirillum  I.  of  Wernicke. — This  organism  is  about 
twice  as  large  as  the  cholera  spirillum,  liquefies  gelatin 
more  rapidly,  produces  indol,  and  is  feebly  pathogenic 
for  guinea-pigs. 

Spirillum  II.  of  Wernicke. — This  spirillum  is  smaller 
than  the  cholera  spirillum,  liquefies  gelatin  more  slowly, 
produces  indol,  and  is  highly  pathogenic  for  rabbits, 
guinea-pigs,  pigeons,  and  mice. 

Spirillum  Bonhoffi. — This  organism  (Fig.  91)  was 
found  in  water  by  BonhofF.  It  has  a  decided  resem- 


N&&&K 

*?&&&& 
%^v 

FIG.  91. — Spirillum  Bonhoffi,  from  a  culture  upon  agar-agar;    x  1000  (Itzerott 
and  Niemann). 

blance  to  the  cholera  spirillum,  but  is  rather  stouter 
and  less  curved.  Curved  forms — i.  e.  semicircles,  sig- 
moids,  and  spirals — occur  in  old  cultures  especially. 

These  organisms  are  colored  badly  with  ordinary  stains, 
dahlia  seeming  to  be  the  most  appropriate  color,  and  ac- 
complishing the  process  better  if  warmed.  The  organ- 
ism is  motile,  and  has  a  long  flagellum  attached  to  one 
end. 

The  colonies  develop  slowly  upon  gelatin  plates,  first 
appearing  in  forty-eight  hours  as  little  grayish  points. 


294  PATHOGENIC  BACTERIA. 

The  margin  of  the  colony  is  sharply  circumscribed  ;  the 
interior  is  broken  up.  The  gelatin  is  not  liquefied.  In 
gelatin  punctures  there  is  no  liquefaction  observable. 

Upon  agar-agar  the  development  at  the  temperature 
of  the  incubator,  which  is  more  rapid  than  that  at  the 
temperature  of  the  room,  results  in  the  production  of  a 
bluish-gray  layer. 

The  growth  upon  potato  has  a  brownish  color.  The 
growth  in  bouillon  and  in  peptone  solutions  is  accompa- 
nied by  the  production  of  indol. 

The  spirillum  is  pathogenic  for  mice,  guinea-pigs,  and 
canary  birds. 

Spirillum  Weibeli. — This  spirillum  (Fig.  92)  was  found 
in  1892  by  Weibel  in  spring-water  which  had  a  long  time 


FIG.  92. — Spirillum  Weibeli,  from  agar-agar;  x  1000  (Itzerott  and  Niemann). 

before  been  infected  by  cholera.  It  is  short,  rather  thick, 
and  distinctly  bent,  often  forming  S-shaped  figures. 

The  colonies  before  liquefaction  sets  in  are  described 
as  pale-brown,  transparent,  circular,  and  homogeneous. 
L/iquefaction  is  much  more  rapid  than  in  cholera,  and 
causes  the  borders  of  the  colonies  to  become  irregular. 
In  the  centre  of  each  colony  a  little  depression  is  ob- 
served. 

In  gelatin  puncture-cultures  the  growth  is  rapid,  be- 


SPIRILLA  RESEMBLING   CHOLERA.  295 

ginning  first  upon  the  surface,  where  a  large  flat,  saucer- 
shaped  liquefaction,  extending  to  the  sides  of  the  tube, 
forms.  Scarcely  any  growth  takes  place  in  the  puncture, 
but  the  superficial  liquefaction,  separated  by  a  horizontal 
line  from  the  normal  gelatin,  descends  slowly. 

Upon  agar-agar  a  grayish- white  layer  is  formed. 

No  growth  has  been  obtained  upon  potato. 

In  alkaline  peptone  solution  a  slow  but  luxuriant 
growth  takes  place. 

Spirillum  Milleri. — This  spirillum  (Fig.  93)  was  found 
in  the  mouth  by  Miller  in  1885.  It  resembles  the  cholera 


FIG.  93. — Spirillum  Milleri,  from  an  agar-agar  culture;    x  1000  (It2erott  and 

Niemann). 

spirillum  somewhat,  but  is  much  more  like  the  spirillum 
of  Finkler  and  Prior,  with  which  many  bacteriologists 
think  it  identical. 

Upon  gelatin  the  colonies  are  small,  finely  granular, 
have  a  narrow  border-zone  and  a  pale-brown  color.  The 
gelatin  is  rapidly  liquefied. 

Upon  agar-agar  a  thick  yellowish  layer  is  produced. 

The  organism  seems  not  to  be  pathogenic. 

Spirillum  Aquatilis. — Giinther  in  1892  found  this  or- 
ganism (Fig.  94)  in  the  water  of  the  river  Spree.  It  is 
similar  to  the  cholera  spirillum  in  shape,  has  a  long 
terminal  flagellum,  and  is  motile. 


296  PATHOGENIC  BACTERIA. 

The  colonies  which  form  upon  gelatin  are  circular, 
have  smooth  borders,  and  look  very  much  as  if  bored  out 
with  a  tool.  They  have  a  brown  color  and  are  finely 


JK 

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v,  *^fMX  IfiSASAfll-  J 


'X  Mmr<Sfs 


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FIG.  94. — Spirillum  aquatilis,  from  an  agar-agar  culture;    x  1000  (Itzerott  and 

Niemann). 

granular.  In  gelatin  puncture-cultures  the  growth  occurs 
almost  exclusively  at  the  surface. 

The  agar-agar  cultures  are  similar  to  those  of  cholera. 

Scarcely  any  development  occurs  in  bouillon.  By  the 
growth  of  the  organism  sulphuretted  hydrogen  gas  is 
produced. 

The  spirillum  does  not  grow  at  all  upon  potato. 

Giinther  did  not  find  the  organism  to  be  pathogenic. 

Spirillum  Terrigenus. — This  species,  also  discovered 
by  Giinther,  was  secured  from  earth.  It  generally  occurs 
in  a  slightly  curved  form,  but  sometimes  is  spiral.  It  is 
actively  motile  and  has  a  terminal  flagellum. 

The  colonies,  which  appear  in  twenty-four  hours,  are 
small,  structureless,  and  transparent,  and  later  take  on  a 
4 '  fat-drop ' '  appearance. 

Upon  agar-agar  a  thin  white  coating  is  formed.  Milk 
is  coagulated  by  the  growth  of  the  organism.  No  indol 
is  produced. 

The  organism  does  not  stain  by  Gram's  method,  and 
is  said  not  to  be  pathogenic  for  guinea-pigs  or  for  mice. 


CHAPTER  VIII. 
PNEUMONIA. 

THE  term  "pneumonia,"  while  generally  understood 
to  refer  to  the  lobar  disease  particularly  designated  as 
croupous  pneumonia,  is  a  vague  one,  really  comprehend- 
ing a  variety  of  conditions  quite  dissimilar  in  character. 
This  being  true,  no  one  will  be  surprised  to  find  that  a 
single  organism  cannot  be  described  as  "specific"  for 
them  all.  Indeed,  pneumonia  must  be  considered  as  a 
group  of  diseases,  and  the  various  microbes  found  asso- 
ciated with  it  must  be  described  successively  in  connection 
with  the  peculiar  phase  of  the  disease  in  which  they  occur. 

i.  Lobar  or  Croupous  Pneumonia. — The  bacterium, 
which  can  be  demonstrated  in  at  least  75  per  cent,  of  the 
cases  of  lobar  pneumonia,  which  is  now  almost  uni- 
versally accepted  as  the  cause  of  the  disease,  and  about 
whose  specificity  very  few  doubts  can  be  raised,  is  the 
pneumococcus  of  Frankel  and  Weichselbaum. 

Priority  of  discovery  in  the  case  of  the  pneumococcus 
seems  to  be  in  favor  of  Sternberg,  who  as  early  as  1880  de- 
scribed an  identical  organism  which  he  secured  from  his 
saliva.  Curiously  enough,  Pasteur  seems  to  have  cap- 
tured the  same  organism,  also  from  saliva,  in  the  same 
year.  The  researches  of  the  observers  whose  names  are 
attached  to  the  organism  were  not  completed  until  five 
years  later.  It  is  to  Frankel,  Telamon,  and  particularly 
to  Weichselbaum,  however,  that  we  are  indebted  for  the 
discovery  of  the  relation  which  the  organism  bears  to 
pneumonia. 

The  pneumococcus  should  rather  be  called  the  pneumo- 
bacillus,  for  it  habitually  has  an  elongated  form,  and  in 
its  most  typical  form  is  so  distinctly  elongate  as  to  be 

297 


298  PATHOGENIC  BACTERIA. 

described  as  lanceolate.  However,  popular  parlance  has 
now  made  it  almost  impossible  to  introduce  Bacillus  pneu- 
monice  instead  of  Diplococcus  pneumonia  (Weichselbaum), 
especially  as  there  is  already  another  organism  bearing 
that  name.  (See  Bacillus  pneumonia  of  Friedlander. ) 
The  organism  (Fig.  95)  is  variable  in  its  morphology. 
When  grown  in  bouillon  it  is  oval,  has  a  pronounced  dis- 


FIG.  95. — Diplococcus  pneumoniae,  from  the  heart's  blood  of  a  rabbit;    x  iooo> 
(Frankel  and  Pfeiffer). 

position  to  occur  in  pairs,  and  not  infrequently  forms 
chains  of  five  or  six  members,  so  that  some  have  been 
disposed  to  look  upon  it  as  a  streptococcus  (Gamaleia). 
In  the  fibrinous  exudate  from  croupous  pneumonia,  in 
the  rusty  sputum,  and  in  the  blood  of  rabbits  and  mice 
containing  them  the  organisms  are  arranged  in  pairs, 
exhibit  a  distinct  lanceolate  shape,  the  pointed  ends 
generally  approximated,  and  are  usually  surrounded  by 
a  distinct  halo  or  capsule  of  clear,  colorless,  homogeneous 
material,  thought  by  some  to  be  a  swollen  cell-wall,  by 


U  IN  I  v  L_  n  o  i  i  T 

OF 


PNEUMONIA.  299 

others  a  mucus-like  secretion  given  off  by  the  cells.  When 
grown  ordinarily  in  culture-media,  and  especially  upon 
solid  media,  the  capsules  are  absent. 

The  organism  is  without  motility,  has  no  spores,  and 
does  not  seem  to  be  able  to  resist  any  unfavorable  con- 
ditions when  grown  artificially.  It  stains  well  with  the 
ordinary  solutions  of  the  anilin  dyes,  and  gives  most 
beautiful  pictures  in  blood  and  tissues  when  stained  by 
Gram's  method.  The  capsule  does  not  stain. 

The  bacillus  is  no  stranger  to  us,  but  can  frequently  be 
found  in  the  saliva  of  healthy  individuals,  and  the  inocu- 
lation of  human  saliva  into  rabbits  generally  causes  a 
septicemia  in  which  the  bacillus  is  found  abundantly  in 
the  blood  and  tissues.  Because  of  its  constant  presence 
in  the  saliva  it  was  described  by  Flugge  as  the  Bacillus 
septicus  sputigenus. 

When  desired  for  purposes  of  study,  it  can  be  obtained 
by  inoculating  rabbits  with  saliva  and  recovering  the  or- 
ganisms from  their  blood,  or  it  can  be  secured  from  the 
rusty  sputum  of  pneumonia  by  the  method  employed 
by  Kitasato  for  securing  tubercle  bacilli  from  sputum. 
A  single  mouthful  of  fresh  sputum  is  secured,  washed 
in  several  changes  of  sterile  water  to  free  it  from  bacteria 
of  the  mouth  and  pharynx,  carefully  separated,  and  a  cen- 
tral portion  transferred  to  an  appropriate  culture-medium. 

The  organism  grows  upon  all  the  culture-media  except 
potato,  but  only  between  the  temperature  extremes  of 
24°  and  42°  C. ;  the  best  development  is  at  37°  C.  The 
growth  is  always  limited,  probably  because  the  formic 
acid  produced  serves  to  check  it.  The  addition  of  an 
unusual  amount  of  alkali  to  the  culture-medium  favors 
the  growth. 

The  organisms  readily  lose  their  virulence  in  culture- 
media,  and  cease  to  be  pathogenic  after  a  few  days.  Not 
only  is  this  true,  but  they  seem  to  be  unable  to  accom- 
modate themselves  to  a  purely  saprophytic  life,  and  un- 
less continually  transplanted  to  new  media  die  in  a  week 
or  two,  sometimes  sooner. 


300  PATHOGENIC  BACTERIA. 

The  colonies  which  develop  at  24°  C.  upon  15  per 
cent,  gelatin  plates  are  described  as  small,  round,  cir- 
cumscribed, finely  granular  white  points  which  grow 
slowly,  never  attain  any  considerable  size,  and  do  not 
liquefy  the  gelatin  (Fig.  96). 


FIG.  96. — Diplococcus  pneumoniae :  colony  twenty-four  hours  old  upon  gelatin ; 
x  100  (Frankel  and  Pfeiffer). 

If,  instead  of  gelatin,  agar-agar  be  used  and  the  plates 
kept  at  the  temperature  of  the  body,  the  colonies  which 
develop  upon  the  plates  appear  as  transparent,  delicate, 
drop-like  accumulations,  scarcely  visible  to  the  naked 
eye,  but  under  the  microscope  distinctly  granular,  the 
central  darker  portion  being  frequently  surrounded  by  a 
paler  marginal  zone. 

In  gelatin  puncture-cultures,  made  with  15  instead  of 
the  usual  10  per  cent,  of  gelatin,  the  growth  takes  place 
along  the  entire  path  of  the  wire  in  the  form  of  little 
whitish  granules  distinctly  separated  from  each  other. 
The  growth  in  gelatin  is  always  very  limited. 

Upon  agar-agar  and  blood-serum  the  growth  consists 
of  minute,  transparent,  semi-confluent,  colorless,  dew- 
drop-like  colonies,  which  die  before  attaining  a  size 


PNEUMONIA.  301 

which  permits  of  their  being  seen  without  careful  in- 
spection. 

In  bouillon  the  organisms  grow  well,  clouding  the 
medium  very  slightly. 

Milk  is  quite  well  adapted  as  a  culture-medium,  its 
casein  being  coagulated. 

No  growth  can  be  secured  upon  potato  at  any  tem- 
perature or  by  any  manipulation  yet  known. 

When  it  is  desired  to  maintain  the  virulence  of  a  cul- 
ture, it  must  be  very  frequently  passed  through  the  body 
of  a  rabbit. 

If  a  small  quantity  of  a  pure  culture  of  the  virulent 
organism  is  introduced  into  a  mouse,  rabbit,  or  guinea- 
pig,  the  animal  dies  in  one  or  two  days.  Exactly  the 
same  result  can  be  obtained  by  the  introduction  of  a 
piece  of  the  lung-tissue  from  croupous  pneumonia,  by 
the  introduction  of  some  of  the  rusty  sputum,  and  gener- 
ally by  the  introduction  of  saliva. 

The  post-mortem  shows  that  an  inflammatory  change 
has  taken  place  at  the  point  of  inoculation,  with  a  fibrin- 
ous  exudate  resembling  somewhat  that  in  diphtheria. 
At  times,  and  especially  in  dogs,  there  may  be  a  little 
pus  formed.  The  other  appearances  are  those  of  a 
general  disturbance.  The  spleen  is  much  enlarged,  is 
firm  and  red  brown.  The  blood  in  all  the  organs  contains 
large  numbers  of  the  bacteria,  most  of  which  exhibit  a 
distinct  lanceolate  form  and  have  their  capsules  very 
distinct.  The  disease  is  a  pure  septicemia  unassociated 
with  pronounced  tissue-changes. 

In  cases  of  the  kind  described  the  lungs  show  no  pneu- 
monic changes.  Likewise,  if  the  hypodermic  needle 
used  for  injection  be  plunged  through  the  breast-wall 
into  the  pulmonary  tissue,  no  pneumonia  results.  Mon- 
ti, however,  claims  to  have  found  that  a  true  character- 
istic pneumonia  results  from  the  injection  of  cultures 
into  the  trachea  of  susceptible  animals.  This  observa- 
tion lacks  confirmation. 

Not  all  animals  are  susceptible.     Guinea-pigs,  mice, 


302  PATHOGENIC  BACTERIA. 

and  rabbits  are  highly  sensitive  to  the  operations  of  the 
organism  ;  dogs  are  comparatively  immune. 

From  this  brief  review  of  the  peculiarities  of  the  pneu- 
mococcus  it  must  be  obvious  that  its  reputation  in  pneu- 
monia depends  more  upon  the  regularity  with  which  it  is 
found  in  that  disease  than  upon  its  capacity  to  produce  a 
similar  affection  in  the  lower  animals. 

As  in  numerous  other  diseases,  we  are  unable  to  furnish 
an  absolute  proof  of  specificity  according  to  the  postu- 
lates of  Koch. 

The  disease  is  peculiar  in  that  recovery  from  it  is  fol- 
lowed either  by  no  immunity  or  by  one  of  such  brief  dura- 
tion as  to  allow  of  frequent  relapses  ;  and  it  is  well  known 
that  many  cases  show  a  subsequent  predisposition  to 
fresh  attacks  of  the  disease.  This  brevity  of  immunity 
lessens  the  probability  that  in  the  future  we  shall  dis- 
cover an  antitoxin  that  shall  be  powerful  in  its  influ- 
ence upon  the  course  and  termination  of  the  disease. 

The  experiments  of  the  Klemperers  showed  that  the 
serum  of  immunized  rabbits  protected  other  animals, 
and  excited  our  interest  a  few  years  ago ;  they,  how- 
ever, failed  when  the  principle  was  applied  in  human 
medicine,  and  the  treatment  of  pneumonia  by  the  in- 
jection of  blood-serum  from  convalescents  has  been 
given  up  as  useless  and  dangerous. 

The  pneumococcus  is  pathogenic  in  other  ways  than 
by  the  production  of  croupous  pneumonia ;  thus,  Foa, 
Bordoni-Uffreduzzi,  and  others  have  found  it  in  cerebro- 
spinal  meningitis  ;  Frankel,  in  pleuritis  ;  Weichselbaum, 
in  peritonitis  ;  Banti,  in  pericarditis  ;  numerous  observers 
have  found  it  in  acute  abscesses ;  Gabbi  has  isolated  it 
from  a  case  of  suppurative  tonsillitis  ;  and  Zaufal,  Levy, 
and  Schrader  and  Netter  have  been  able  to  demonstrate 
its  presence  in  the  pus  of  otitis  media.  It  has  also  been 
reported  as  occurring  in  the  joints  in  arthritis  following 
pneumonia. 

The  pneumococcus  no  doubt  is  habitually  present  in 
the  mouth  of  almost  every  healthy  person.  Its  entrance 


PNEUMONIA.  303 

into  the  lung  is  therefore  only  a  matter  of  accident,  and 
an  unusually  long  sigh,  a  deep  inspiration  before  a  cough 
or  sneeze,  or  some  unusual  respiratory  movement,  serves 
to  draw  it  into  the  bronchioles,  which  are  normally  free 
from  bacteria. 

In  the  opinion  of  most  authorities,  something  more 
than  the  simple  entrance  of  the  bacterium  into  the  lung 
is  required  for  the  production  of  the  disease,  but  what 
that  something  is,  is  still  a  matter  of  doubt.  It  would 
seem  to  be  some  systemic  depravity,  and  in  support  of  this 
view  we  may  point  out  that  pneumonia  is  very  frequent, 
and  almost  universally  fatal,  among  drunkards.  Whether, 
however,  any  vital  depression  or  systemic  depravity  will 
predispose  to  the  disease,  or  whether  it  depends  for  its 
origin  upon  the  presence  of  a  certain  leucomaine,  time 
and  further  study  will  be  required  to  tell. 

Bacillus  Pneumonia  of  Friedldnder  (Fig.  97). — An  un- 


FIG.  97. — Bacillus  pneumoniae  of   Friedlander,  from  the  expectoration  of  a 
pneumonia  patient;  x  1000  (Frankel  and  Pfeiffer). 

fortunate  accident  has  applied  the  name  "pneumococcus" 
to  an  organism  very  different  from  the  one  just  described. 
It  was  discovered  by  Friedlander  in  1883  in  the  exudate 
from  the  lung  in  croupous  pneumonia,  and,  being  thought 


304  PA  THOGENIC  BA  CTERIA . 

by  its  discoverer  to  be  the  cause  of  the  disease,  very  natu- 
rally was  called  the  pneumococcus,  or,  more  correctly,  the 
pneumobacillus.  The  grounds  upon  which  the  pathog- 
eny  of  the  organism  was  supposed  to  depend  were  very  in- 
sufficient, and  the  bacillus  of  Friedlander — or,  as  Fliigge 
prefers  to  call  it,  the  Bacillus  pneumonise — has  ceased  to 
be  regarded  as  specific,  and  is  now  looked  upon  as  an 
accidental  organism  whose  presence  in  the  lung  is,  in 
most  cases,  unimportant. 

As  the  two  organisms  are  similar  in  more  respects  than 
their  names,  Friedlander' s  bacillus  requires  at  least  a 
brief  description. 

It  is  distinctly  a  bacillus,  but  sometimes,  when  occur- 
ring in  pairs,  has  a  close  resemblance  to  the  pneumo- 
coccus of  Frankel  and  Weichselbaum.  Very  frequently 
it  forms  chains  of  four  or  more  elements.  It  is  also  com- 
monly surrounded  by  a  transparent  capsule.  It  is  non- 
motile,  has  no  spores  and  no  flagella.  It  stains  well 
with  the  ordinary  anilin  dyes,  but  does  not  retain  the 
color  when  stained  by  Gram's  method. 

Frankel  points  out  that  Friedlander' s  error  in  suppos- 
ing this  bacillus  to  be  the  chief  parasite  in  pneumonia 
depended  upon  the  fact  that  his  studies  were  made  by 
the  plate  method.  If  some  of  the  pneumonic  exudate  be 
mixed  with  gelatin  and  poured  upon  plates,  the  bacilli 
grow  into  colonies  at  the  end  of  twenty-four  hours,  and 
appear  as  small  white  spheres  which  spread  upon  the 
gelatin  to  form  white  masses  of  a  considerable  size. 
Under  the  microscope  these  colonies  are  rather  irregular 
in  outline  and  somewhat  granular. 

The  bacillus  grows  at  as  low  a  temperature  as  16°  C., 
and,  according  to  Sternberg,  has  a  thermal  death-point 
of  56°  C. 

When  a  colony  is  transferred  to  a  gelatin  puncture-cul- 
ture, quite  a  massive  growth  occurs.  Upon  the  surface  a 
somewhat  elevated,  rounded  white  mass  is  formed,  and 
in  the  track  of  the  wire  innumerable  little  colonies 
spring  up  and  become  confluent,  so  that  a  u  nail-growth  " 


PNEUMONIA.  305 

results.  No  liquefaction  occurs.  When  old  the  cultures 
sometimes  become  brown  in  color. 

Upon  the  surface  of  agar-agar  at  ordinary  temperatures 
quite  a  luxuriant  white  or  brownish-yellow,  smeary,  cir- 
cumscribed growth  occurs.  The  growth  upon  blood- 
serum  .  is  the  same. 

Upon  potato  the  growth  is  abundant,  quickly  covering 
the  entire  surface  with  a  thick  yellowish-white  layer, 
which  sometimes  contains  bubbles  of  gas.  Gas  is  also 
sometimes  developed  in  gelatin  cultures. 

A  most  superficial  comparison  will  suffice  to  show  the 
great  difference  in  vegetation  between  these  two  so-called 
pneuinococci. 

Friedlander  had  considerable  difficulty  in  causing  any 
pathogenic  changes  by  the  injection  of  his  bacillus  into 
animals.  Rabbits  and  guinea-pigs  were  immune,  and 
the  only  actual  pathogenic  results  which  Friedlander  ob- 
tained were  in  mice,  into  whose  lungs  and  pleura  he 
injected  the  cultures.  The  remarks  of  Frankel  upon 
such  mouse-operations,  which  do  not  add  much  weight 
to  experiments,  have  already  been  quoted. 

In  the  status  prczsens  of  bacteriologic  knowledge  the 
bacillus  of  Friedlander  is  regarded  as  an  organism  of  very 
feeble  pathogenic  powers,  generally  a  harmless  sapro- 
phyte, but  which  may  at  times  aid  in  producing  inflam- 
matory changes  when  in  the  tissues  of  the  human  body. 

2.  Catarrhal  Pneumonia. — This  form  of  pulmonary 
inflammation   occurs   in   local   areas,   generally  situated 
about   the   distribution  of  a  bronchiole.     It   cannot   be 
said  to  have  a  specific   micro-organism,  as   almost   any 
irritant  foreign  materials  accidentally  inhaled  can  cause 
it.     The  majority  of  the  cases,  however — and  especially 
those  which  are  distinctly  peribronchial — are  caused  by 
the  presence  of  the  staphylococcus  and  streptococcus  of 
suppuration.     Friedlander' s  bacillus  may  also  aid  in  pro- 
ducing local  inflammations. 

3.  Tubercular  Pneumonia. — At  times  the  process  of 
pulmonary  tuberculosis  is  so  rapid,  and  associated  with 

20 


306  PATHOGENIC  BACTERIA. 

the  production  of  so  much  semi-liquid,  semi-necrotic 
material,  that  the  auto-infection  of  the  lung  is  greatly 
favored  ;  the  tubercle  bacilli  are  distributed  to  the  entire 
lung  or  to  large  parts  of  it,  and  a  distinct  inflammation 
occurs.  Such  a  pneumonia  may  be  caused  by  the  tubercle 
bacillus  alone,  but  more  often  it  is  aided  by  accompany- 
ing staphylococci,  streptococci,  tetragenococci,  pneumo- 
cocci,  pneumobacilli,  and  other  organisms  apt  to  be  pres- 
ent in  a  lung  in  which  tuberculosis  is  in  progress  and 
ulceration  and  cavity-formation  are  advanced. 

4.  Mixed  Pneumonias. — It  frequently  happens  that 
pneumonia  occurs  in  the  course  of,  or  shortly  after  the 
convalescence  from,  influenza.  In  these  cases  a  mixed 
infection  is  present,  and  there  is  no  difficulty  in  deter- 
mining that  both  the  influenza  bacillus  and  the  pneumo- 
coccus  are  present.  Again,  sometimes  the  pneumococci 
and  staphylococci  operate  simultaneously,  and  produce 
a  purulent  pneumonia  with  abscesses  as  the  conspicuous 
feature.  As  almost  any  combination  of  the  described 
bacteria  is  possible  in  the  lungs,  and  as  these  combi- 
nations will  all  produce  varying  inflammatory  conditions, 
it  must  be  left  for  the  student  to  imagine  what  the  par- 
ticular characters  of  each  may  be. 

Among  these  mixed  pneumonias  may  be  mentioned 
those  called  by  Klemperer  and  Levy  "complicating 
pneumonias,"  occurring  in  the  course  of  typhoid, .  etc. 


C.     THE  SEPTIC  DISEASES. 


CHAPTER   I. 
RELAPSING   FEVER. 

As  long  ago  as  1873,  Obermeier  discovered  that  a 
flexible  spiral  organism,  about  o.i  //  in  diameter  and 
from  20-40  p.  in  length,  could  be  observed  in  the  blood 
of  patients  suffering  from  relapsing  fever. 

Although  many  of  the  best  bacteriologists  of  our  day 
have  occupied  themselves  with  the  study  of  this  spiril- 
lum, we  really  have,  at  present,  very  little  more  know- 
ledge than  that  given  us  by  Obermeier. 


• 


>  *' 

*    ®  " 


FIG.  98.  —  Spirochseta  febris  recurrentis;   x  650  (Heim). 

The  spirilla  (Fig.  98)  are  generally  very  numerous, 
are  long,  slender,  and  flexible  (spirochaeta),  and  possess 
a  vigorous  movement  by  flagella.  The  ends  are  rather 
pointed. 

The  spirillum  stains  well  by  ordinary  methods,  but 

307 


308  PATHOGENIC  BACTERIA. 

not  by  Gram's  method.  It  seems  to  be  a  strict  parasite, 
and  has  never  been  cultivated  artificially. 

Of  the  pathogenesis  of  the  organism  there  can  be  no 
doubt,  as  it  is  invariably  present  in  relapsing  fever  and 
undergoes  a  peculiar  cycle  of  changes  according  to  the 
stage  of  the  disease.  During  the  pyrexia  the  organisms 
are  found  in  the  blood  in  active  movement,  swimming 
both  by  rotation  on  the  long  axis  and  by  undulation. 
As  soon  as  the  crisis  comes  on  they  are  found  to  be  with- 
out motion,  most  of  them  enclosed  in  leucocytes  and 
seemingly  dead.  The  recurrence  of  the  paroxysm  has 
suggested  to  many  that  spores  are  formed  in  the  spiril- 
lum, but  no  one  has  been  successful  in  proving  that  this 
is  the  case.  Koch,  Carter,  and  Soudakewitch  have  all 
succeeded  in  giving  the  disease  to  monkeys,  and  Munch 
and  Moczutkowsky  have  gone  further  and  have  produced 
it  in  men  by  introducing  into  them  blood  from  diseased 
patients. 

Soudakewitch  finds  that  the  removal  of  the  spleen 
causes  the  disease  to  terminate  fatally  in  monkeys. 


CHAPTER   II. 

INFLUENZA. 

NOTWITHSTANDING  a  large  number  of  bacteriologic 
examinations  conducted  for  the  purpose  of  determining 
the  cause  of  influenza,  it  was  not  until  1892,  after  the 
great  epidemic,  that  there  was  found  simultaneously  by 
Canon  and  PfeifFer  a  bacterium  which  conformed,  at  least 
in  large  part,  to  the  requirements  of  specificity. 

The  observers  mentioned  found  the  same  organism — 
one  in  the  blood  of  influenza  patients,  the  other  in  the 
purulent  bronchial  discharges. 

The  specific  organisms  (Fig.  99)  are  bacilli,  very  small 
in  size,  having  about  the  same  diameter  as  the  bacillus 


Jh     * 

1      '***  '«' 


'V^v'-'  tf?V- 


FIG.  99. — Bacillus  influenzae,  from  a  gelatin  culture;    x  1000  (Itzerott  and 

Niemann). 

of  mouse-septicemia,  but  only  about  half  as  long  (o.  2  by 
0.5  //).  They  are  usually  solitary,  but  may  be  united  in 
chains  of  three  or  four  elements.  They  stain  rather 

309 


310  PATHOGENIC  BACTERIA. 

poorly,  except  with  such  concentrated  penetrating  stains 
as  carbol-fuchsin  and  L,6ffler's  alkaline  methylene  blue, 
and  even  with  these  the  bacilli  stain  more  deeply  at  the 
ends  than  in  the  middle,  so  that  they  appear  not  a  little 
like  diplococci. 

For  the  demonstration  of  the  bacilli  in  the  blood  Canon 
recommends  a  rather  complicated  method.  The  blood  is 
spread  upon  clean  cover-glasses  in  the  usual  way,  thor- 
oughly dried,  and  then  fixed  by  immersion  in  absolute 
alcohol  for  five  minutes.  The  stain  which  seems  best  is 
Czenzynke's : 

Concentrated  aqueous  solution  of  methylene 

blue,  40 ; 

0.5  per  cent,  solution  of  eosin  in  70  per  cent. 

alcohol,  20 ; 

Distilled  water,  40. 

The  cover-glasses  are  immersed  in  this  solution,  and  kept 
in  the  incubator  for  three  to  six  hours,  after  which  they 
are  washed  in  water,  dried,  and  then  mounted  in  Canada 
balsam.  By  this  method  the  erythrocytes  are  stained  red, 
the  leucocytes  blue,  and  the  bacillus,  which  is  also  blue, 
appears  as  a  short  rod  or  often  as  a  dumb-bell. 

Sometimes  large  numbers  of  the  bacilli  are  present ; 
sometimes  very  few  can  be  found  after  prolonged  search. 
They  are  often  enclosed  within  the  leucocytes.  It  really 
is  not  necessary  to  pursue  so  tedious  a  staining  method 
for  demonstrating  the  bacilli,  for  they  stain  quite  well  by 
ordinary  methods.  They  do  not  stain  by  Gram's  method. 

The  bacillus  is  non-motile,  and,  so  far  as  is  known, 
does  not  form  spores.  Its  resisting  powers  are  very  re- 
stricted, as  it  speedily  succumbs  to  drying,  and  is  cer- 
tainly killed  by  an  exposure  to  a  temperature  of  60°  C. 
for  five  minutes.  It  will  not  grow  at  any  temperature 
below  28°  C. 

The  bacillus  does  not  grow  in  gelatin  or  upon  ordinary 
agar-agar.  Upon  glycerin  agar-agar,  after  twenty-four 
hours  in  the  incubator,  minute  colorless,  transparent, 


INFLUENZA.  311 

drop-like  cultures  may  be  seen  along  the  line  of  inocula- 
tion. They  do  not  look  unlike  condensed  moisture,  and 
Kitasato  makes  a  special  point  of  the  fact  that  the  colo- 
nies never  become  confluent.  The  colonies  may  at  times 
be  so  small  as  to  require  a  lens  for  their  discovery. 

In  bouillon  a  scant  development  occurs,  small  whitish 
particles  appearing  upon  the  surface,  subsequently  sink- 
ing to  the  bottom  and  causing  a  "woolly"  deposit  there. 
While  the  growth  is  so  delicate  in  these  ordinary  media, 
the  bacillus  grows  quite  well  upon  culture-media  contain- 
ing hemoglobin  or  blood,  and  can  be  transferred  from 
culture  to  culture  many  times  before  it  loses  its  vitality. 

It  cannot  be  positively  proven  that  this  bacillus  is  the 
cause  of  influenza,  but  from  the  fact  that  the  bacillus 
can  be  found  only  in  cases  of  influenza,  that  its  presence 
corresponds  with  the  course  of  the  disease  in  that  it  is 
present  as  long  as  the  purulent  secretions  last,  and  then 
disappears,  and  that  PfeifFer  was  able  to  demonstrate  its 
presence  in  all  cases  of  uncomplicated  influenza,  his  con- 
clusion that  the  bacillus  is  specific  is  certainly  justifiable. 


CHAPTER   III. 
MALIGNANT   EDEMA. 

THE  chief  contaminating  organism  in  the  preparation 
of  pure  cultures  of  the  tetanus  bacillus  is  a  large  slender 
bacillus  almost  as  large  as  that  of  anthrax,  but  with 
rounded  ends  and  an  individual  motility  accomplished 
by  means  of  flagella  attached  to  its  ends  and  sides 
(Fig.  100).  It  is  a  strictly  anaerobic  bacterium,  and  was 


FIG.  100. — Bacillus  of  malignant  edema,  from  the  body-juice  of  a  guinea-pig 
inoculated  with  garden-earth;    x  1000  (Frankel  and  Pfeiffer). 

originally  described  by  Pasteur  (1875)  as  the  Vibrion 
septique.  It  grows  well  at  the  room-temperature,  as  well 
as  at  the  temperature  of  the  incubator,  produces  oval 
central  spores,  and,  because  of  its  association  with  a  spe- 
cific edema  in  certain  animals,  is  known  as  the  Bacillus 
oedema  maligni. 

312 


MALIGNANT  EDEMA.  313 

The  organism  is  widely  distributed  in  nature,  being 
almost  always  present  in  garden-earth.  It  is  also  found 
in  various  dusts,  in  the  waste  water  from  houses,  and 
sometimes  in  the  intestinal  canals  of  animals. 

When  introduced  beneath  the  skin  this  bacillus  proves 
pathogenic  for  a  large  number  of  animals — mice,  guinea- 
pigs,  rabbits,  horses,  dogs,  sheep,  goats,  pigs,  calves, 
chickens,  and  pigeons.  Cattle  seem  to  be  immune. 

Gunther  points  out  that  the  simple  inoculation  of  the 
bacillus  upon  an  abraded  surface  is  insufficient  to  pro- 
duce the  disease,  because  the  oxygen  which  is,  of  course, 
abundant  there  is  detrimental  to  its  growth.  When  an 
experimental  inoculation  is  performed,  a  small  subcu- 
taneous pocket  should  be  made,  and  the  bacilli  introduced 
into  it  in  such  a  manner  as  not  to  be  in  contact  with  the 
air. 

If  the  inoculated  animal  be  a  mouse,  guinea-pig,  or 
rabbit,  in  about  forty-eight  hours  it  sickens  and  dies. 
The  autopsy  shows  a  general  subcutaneous  edema  con- 
taining immense  numbers  of  the  bacilli.  In  the  blood 
the  bacilli  are  few  or  cannot  be  found,  because  of  the 
oxygen  which  it  contains.  The  great  majority  of  them 
occupy  the  subcutaneous  tissue,  where  very  little  oxygen 
is  present  and  the  conditions  of  growth  are  therefore  good. 
If  the  animal  is  allowed  to  remain  undisturbed  for  some 
time  after  death,  the  bacilli  spread  to  the  circulatory  sys- 
tem and  reach  all  the  organs. 

Brieger  and  Ehrlich  have  reported  two  cases  of  malig- 
nant edema  in  man.  Both  cases  were  typhoid-fever 
patients  injected  with  musk,  and  developed  the  edema 
in  consequence  of  impurity  of  the  therapeutic  agent. 
No  case  is  reported,  however,  in  which  healthy  men 
have  been  infected  with  the  disease. 

Cornevin  declares  that  the  passage  of  the  bacillus 
through  the  white  rat  diminishes  its  virulence,  and  that 
the  animals  of  various  species  that  recover  from  this 
milder  affection  are  subsequently  immune  to  the  virulent 
organisms. 


PATHOGENIC  BACTERIA. 


The  bacillus  of  malignant  edema  stains  well  with  ordi- 
nary cold  aqueous  solutions  of 
the  anilin  dyes,  but  not  by 
Grain's  method. 

The  organism  is  not  a  difficult 
one  to  secure  in  pure  culture, 
as  has  been  said,  generally  con- 
taminating tetanus  cultures  and 
being  much  more  easy  to  se- 
cure by  itself  than  its  congener. 
It  is  most  easily  obtained  from 
the  edematous  tissues  of  guinea- 
pigs  and  rabbits  inoculated  with 
garden-earth. 

The  colonies  which  develop 
upon  the  surface  of  gelatin  kept 
free  of  oxygen  appear  to  the 
naked  eye  as  small  shining 
bodies  with  liquid  grayish-white 
contents.  They  gradually  in- 
crease in  circumference,  but  do 
not  change  their  appearance. 
Under  the  microscope  they  ap- 
pear filled  with  a  tangled  mass 
of  long  filaments  which  under  a 
high  power  exhibit  individual 
movement.  The  edges  of  the 
colony  have  a  fringed  appearance,  much  like  the  hay  or 
potato  bacillus. 

In  gelatin  tube-cultures  the  characteristic  growth  can- 
not, be  observed  in  a  puncture,  because  of  the  air  which 
remains  in  the  path  of  the  wire.  The  best  preparation 
is  made  by  heating  the  gelatin  to  expel  the  air  it  may 
contain,  inoculating  while  still  liquid,  then  replacing  the 
air  by  hydrogen,  and  sealing  the  tube.  In  such  a  tube 
the  bacilli  develop  near  the  bottom.  The  appearance  of 
the  growth  is  highly  typical,  as  globular  circumscribed 
areas  of  cloudy  liquefaction  result  (Fig.  101),  and  may  con- 


FIG.  101. — Bacillus  of  malig- 
nant edema  growing  in  glucose 
gelatin  (Frankel  and  Pfeiffer). 


MALIGNANT  EDEMA.  315 

tain  a  small  amount  of  gas.  In  gelatin  to  which  a  little 
grape-sugar  has  been  added  the  gas-production  is  marked. 
The  gas  is  partly  inflammable,  partly  CO2.  A  distinct 
odor  accompanies  the  gas-production,  and  is  especially 
noticeable  in  agar-agar  cultures. 


CHAPTER  IV. 
MEASLES. 

IN  1892,  Canon  and  Pielicke,  after  the  investigation  of 
fourteen  cases  of  measles,  reported  the  discovery  of  a 
specific  bacillus  in  the  blood  in  that  disease. 

The  organism  is  quite  variable  in  size,  sometimes 
being  quite  small  and  resembling  a  diplococcus,  some- 
times larger,  and  occasionally  quite  long,  so  that  one 
bacillus  may  be  as  long  as  the  diameter  of  a  red  blood- 
corpuscle. 

The  discovery  was  made  by  means  of  a  peculiar  method 
of  staining,  as  follows :  The  blood  is  spread  in  a  very 
thin,  even  layer  upon  perfectly  clean  cover-glasses,  and 
fixed  by  five  to  ten  minutes'  immersion  in  absolute  alco- 
hol. These  glasses  are  then  placed  in  a  stain  consisting 
of 

Concentrated  aqueous  solution  of  methylene  blue,  40 ; 
0.25  per  ct.  solution  of  eosin  in  70  per  ct.  alcohol,  20  ; 
Distilled  water,  40, 

and  stood  in  the  incubator  at  37°  C.  for  from  six  to 
twenty-four  hours.  The  bacilli  do  not  all  stain  uni- 
formly. 

The  discoverers  of  the  bacillus  claim  to  have  made  it 
grow  several  times  in  bouillon,  but  failed  to  induce  a 
growth  upon  other  media. 

The  bacilli  do  not  stain  by  Gram's  method  ;  they  seem 
to  have  motility ;  no  spores  were  observed.  They  were 
found  not  only  in  the  blood,  but  also  in  the  secretions 
from  the  nose  and  eyes.  They  are  said  to  persist  through- 
out the  whole  course  of  the  disease,  even  occasionally 
being  found  after  the  fever  subsides. 

316 


MEASLES.  317 

Czajrowski  asserts  that  the  bacillus  can  be  cultivated 
upon  various  albuminous  media  except  gelatin  and  agar. 
On  glycerin  agar-agar,  especially  with  the  addition  of 
hemotogen,  and  on  blood-serum,  they  should  grow  in 
three  or  four  days  with  an  appearance  like  that  of  dew- 
drops.  Under  the  microscope  the  colonies  are  structure- 
less. Mice  die  of  a  septicemia  after  a  subcutaneous  in- 
oculation. 


CHAPTER   V. 
BUBONIC  PLAGUE. 

THE  bacillus  of  bubonic  plague  (Fig.   102)  seems  to 
have   met   an   independent   discovery   at   the  hands   of 


FIG.  1 02. — Bacillus  of  bubonic  plague  (Yersin). 

Yersin  and  Kitasato  in  the  summer  of  1894,  during  the 
activity  of  the  plague  then  raging  at  Hong-Kong.  There 
seems  to  be  not  the  slightest  doubt  that  the  micro-organ- 
isms described  by  the  two  observers  are  identical. 

The  bubonic  plague  is  an  extremely  fatal  infectious 
disease,  whose  ravages  in  the  hospital  in  which  Yersin 
made  his  observations  carried  off  95  per  cent,  of  the 
cases.  It  affects  both  men  and  animals,  and  is  character- 
ized by  sudden  onset,  high  fever,  prostration,  delirium, 
and  the  occurrence  of  lymphatic  swellings — buboes — 
affecting  chiefly  the  inguinal  glands,  though  not  infre- 
quently the  axillary,  and  sometimes  the  cervical,  glands. 
Death  comes  on  in  severe  cases  in  forty-eight  hours.  If 
the  case  is  of  longer  duration,  the  prognosis  is  said  to  be 

318 


BUBONIC  PLAGUE.  319 

better.  Autopsy  in  fatal  cases  reveals  the  enlargement 
of  the  lymphatic  glands,  whose  contents  are  soft  and 
sometimes  purulent. 

The  studies  of  Kitasato  and  Yersin  showed  that  in 
blood  drawn  from  the  finger-tips  and  in  the  softened  con- 
tents of  the  glands  a  small  bacillus  was  demonstrable. 
The  organisms  are  small,  stain  much  more  distinctly  at 
the  ends  than  in  the  middle,  so  that  they  resemble 
diplococci,  and  in  fresh  specimens  seem  to  be  surrounded 
by  a  capsule.  Kitasato  compares  the  organism  to  the 
well-known  bacillus  of  chicken-cholera.  It  is  feebly 
motile,  and  does  not  seem  to  form  spores.  Nothing 
is  said  about  the  presence  of  flagella. 

When  cultures  are  made  from  the  softened  contents  of 
the  buboes,  the  bacillus  can  be  obtained  almost  or  quite 
pure,  and  is  found  to  develop  upon  artificial  culture- 
media.  In  bouillon  a  diffuse  cloudiness  results  from 
the  growth,  as  observed  by  Kitasato,  though  in  Yersin' s 
observations  the  cultures  more  nearly  resembled  erysipe- 
las cocci,  and  contained  zooglea  attached  to  the  sides  and 
in  the  bottom  of  a  tube  of  nearly  clear  fluid. 

In  gelatin  puncture-cultures  the  development  is  scant. 
The  medium  is  not  liquefied  (?) ;  the  growth  takes  place 
in  the  form  of  a  fine  duct,  little  points  being  seen  on  the 
surface  and  in  the  line  of  puncture. 

Upon  agar-agar — glycerin  agar-agar  is  best — the  bacilli 
grow  freely,  the  colonies  being  whitish  in  color,  with  a 
bluish  tint  by  reflected  light.  Under  the  microscope 
they  appear  moist,  with  rounded,  uneven  edges.  The 
small  colonies  are  said  to  resemble  little  tufts  of  glass- 
wool  ;  the  larger  ones  have  large  round  centres.  Micro- 
scopic examination  of  the  bacilli  grown  upon  agar- 
agar  reveals  the  presence  of  long  chains  resembling 
streptococci. 

Upon  blood-serum  the  growth  at  the  temperature  of 
the  incubator  is  luxuriant.  It  forms  a  moist  layer  of  a 
yellowish-gray  color,  and  is  unaccompanied  by  liquefac- 
tion of  the  serum. 


320  PATHOGENIC  BACTERIA. 

Upon  potatoes  no  growth  occurs  at  ordinary  tempera- 
tures. When  the  potato  is  stood  away  for  a  few  days  in 
the  incubator,  a  scanty,  dry,  whitish  layer  develops. 

Kitasato  found  that  mice,  rats,  guinea-pigs,  and  rabbits 
are  all  susceptible.  When  blood,  lymphatic  pulp,  or  pure 
cultures  are  inoculated  into  them,  the  animals  become  ill 
in  from  one  to  two  days,  according  to  size.  Their  eyes 
become  watery,  they  begin  to  show  disinclination  to  take 
food  or  to  make  any  bodily  effort,  the  temperature  rises 
to  41.5°  C.,  they  remain  quietly  in  a  corner  of  the  cage, 
and  die  with  convulsive  symptoms  in  from  two  to  five 
days. 

According  to  Yersin,  an  infiltration  can  be  observed 
in  a  few  hours  about  the  point  of  inoculation.  The 
autopsy  shows  the  infiltration  to  be  made  up  of  a  yellow- 
ish gelatinous  exudation.  The  spleen  and  liver  are  en- 
larged, the  former  often  presenting  an  appearance  much 
like  an  eruption  of  miliary  tubercles.  Sometimes  there 
is  universal  swelling  of  the  lymphatic  glands.  Bacilli  are 
found  in  the  blood  and  in  all  the  internal  organs.  Very 
often  there  are  petechial  eruptions  during  life,  and  upon 
the  inner  abdominal  walls  there  are  occasional  hemor- 
rhages. The  intestine  is  hyperemic,  the  adrenals  con- 
gested. There  are  often  sero-sanguinolent  effusions  into- 
the  serous  cavities. 

Kitasato  found  that  pigeons  were  not  susceptible. 
Animals  fed  upon  cultures  or  upon  the  meat  of  others 
dead  of  the  disease  became  ill  and  died  with  typical 
symptoms.  When  he  inoculated  animals  with  the  dust 
of  dwelling-houses  in  which  the  disease  had  occurred, 
some  died  of  tetanus,  one  from  plague.  Many  rats  and 
mice  in  which  examination  showed  the  characteristic 
bacilli  died  spontaneously  in  Hong-Kong. 

Yersin  showed  that  flies  also  die  of  the  disease.  Mace- 
rating and  crushing  a  fly  in  bouillon,  he  not  only  suc- 
ceeded in  obtaining  the  bacillus  from  the  medium,  but 
infected  an  animal  with  it. 

Yersin  found  that  when  cultivated  for  any  length  of 


BUBONIC  PLAGUE.  321 

time  upon  culture-media,  especially  agar-agar,  the  viru- 
lence was  rapidly  lost  and  the  bacillus  eventually  died. 
On  the  other  hand,  when  constantly  inoculated  from 
animal  to  animal  the  virulence  of  the  bacillus  is  much 
increased. 

The  bacillus  probably  attenuates  readily.  Kitasato 
found  that  it  did  not  seem  able  to  withstand  desicca- 
tion longer  than  four  days ;  and  Yersin  found  that  al- 
though it  could  be  secured  from  the  soil  beneath  an 
infected  house  at  a  depth  of  4-5  c.cm.,  the  virulence 
of  such  bacilli  was  lost. 

Kitasato  found  that  the  bacillus  was  killed  by  two 
hours'  exposure  to  0.5  per  cent,  carbolic  acid,  and  also 
by  exposure  to  a  temperature  of  80°  C. 

It  seems  possible  to  make  a  diagnosis  of  the  disease  in 
doubtful  cases  by  examining  the  blood,  but  it  is  admitted 
that  a  good  deal  of  bacteriologic  practice  is  necessary  for 
the  purpose. 

Kitasato' s  experiments  have  shown  that  it  is  possible 
to  bring  about  immunity  to  the  disease,  though  nothing 
definite  in  the  way  of  experiment  has  as  yet  been  re- 
corded. 

21 


CHAPTER   VI. 
TKTRAGENUS. 

THERE  can  sometimes  be  found  in  the  normal  saliva, 
more  commonly  in  tuberculous  sputum,  and  still  more 
commgnly  in  the  cavities  of  tuberculosis  pulmonalis,  a 
large  micrococcus  grouped  in  fours  and  known  as  the 
Micrococcus  tetragenus  (Fig.  103).  It  was  discovered  by 


^^••B 
FIG.  103. — Micrococcus  tetragenus  in  pus  from  a  white  mouse;  x  615  (Heim). 

Gaffky,  and  subsequently  carefully  studied  by  Koch  and 
Gaffky.  It  sometimes  occurs  in  the  pus  of  acute  ab- 
scesses, and  may  be  of  importance  in  connection  with 
the  pulmonary  abscesses  which  so  often  complicate  tu- 
berculosis. 

The  cocci  are  rather  large,  measuring  about  i  //  in 
diameter.  In  cultures  they  show  no  particular  arrange- 
ment among  themselves,  but  in  the  blood  and  tissues  of 
animals  they  commonly  appear  arranged  in  groups  of 
four  surrounded  by  a  transparent  gelatinous  capsule. 

The  organism  stains  well  by  ordinary  methods,  and 

322 


TETRAGENUS.  323 

most  beautifully  by  Gram's  method,  by  which  it  can  be 
best  demonstrated  in  tissues. 

Upon  gelatin  plates  small  white  colonies  are  produced 
in  from  twenty-four  to  forty-eight  hours.  Under  the 
microscope  they  are  found  to  be  spherical  or  elongate 
(lemon-shaped),  finely  granular,  and  lobulated  like  a 
raspberry  or  a  mulberry.  When  superficial  they  form 
white,  elevated,  rather  thick  masses  1-2  mm.  in  diameter 
(Fig.  104). 

In    gelatin    punctures  a  large  white  surface-growth 


.x. 


w 


FIG.  104. — Micrococcus  tetragenus:  colony  twenty-four  hours  old  upon  the  sur- 
face of  an  agar-agar  plate;  x  100  (Heim). 

takes  place,  but  very  scant  development  occurs  in  the 
puncture,  where  the  small  spherical  colonies  generally 
remain  isolated. 

Upon  the  surface  of  agar-agar  spherical  white  colonies 
are  produced.  They  may  remain  isolated  or  may  become 
confluent. 

Upon  potato  a  luxuriant  thick,  white  growth  occurs. 

The  growth  upon  blood-serum  is  also  abundant,  espe- 
cially at  the  temperature  of  the  incubator.  It  has  no 
distinctive  peculiarities. 

The  introduction  of  tuberculous  sputum  or  of  a  most 
minute  quantity  of  a  pure  culture  of  this  coccus  into 
white  mice  generally  causes  a  fatal  septicemia. 


324  PATHOGENIC  BACTERIA. 

The  organisms  are  found  in  small  numbers  in  the 
heart's  blood,  but  are  numerous  in  the  spleen,  lungs, 
liver,  and  kidneys. 

House-mice  and  field-mice  are  comparatively  immune  ; 
dogs  and  rabbits  are  also  highly  resistant.  Guinea-pigs 
sometimes  die  from  general  infection,  though  sometimes 
local  abscesses  may  be  the  only  result  of  subcutaneous 
inoculation. 

The  tetragenococci  are  of  no  special  importance  in 
human  pathology,  but  probably  hasten  the  tissue-necrosis 
in  tuberculosis  pulmonalis,  and  may  aid  in  the  formation 
of  abscesses  of  the  lung  and  contribute  to  the  production 
of  the  hectic  fever. 


CHAPTER  VII. 
CHICKEN- CHOLERA. 

THE  barnyards  of  Europe,  and  sometimes  of  America, 
are  occasionally  visited  by  an  epidemic  disease  which 
affects  pigeons,  turkeys,  chickens,  ducks,  and  geese,  and 
causes  almost  as  much  destruction  among  them  as  the 
occasional  epidemics  of  cholera  and  small-pox  produce 
among  men.  Rabbit-warrens  are  also  at  times  seriously 
affected  by  the  epidemic.  When  fowls  are  ill  with  the 
disease,  they  fall  into  a  condition  of  weakness  and  apathy 
which  causes  them  to  remain  quiet,  seemingly  almost 
paralyzed,  and  ruffle  up  the  feathers.  The  eyes  are 
closed  shortly  after  the  illness  begins,  and  the  birds 
gradually  fall  into  a  stupor  from  which  they  do  not 
awaken.  The  disease  leads  to  a  fatal  termination  in 
twenty-four  to  forty-eight  hours.  During  its  course 
there  is  profuse  diarrhea,  the  very  frequent  fluid,  slimy, 
grayish-white  discharges  containing  numerous  micro- 
organisms. 

The  bacilli  which  are  responsible  for  this  disease  were 
first  observed  by  Perroncito  in  1878,  and  afterward  thor- 
oughly studied  by  Pasteur.  They  are  short,  broad  bacilli 
with  rounded  ends,  sometimes  united  to  each  other, 
with  the  production  of  moderately  long  chains  (Fig.  105). 
Pasteur  at  first  regarded  them  as  cocci,  because  when 
stained  with  a  penetrating  anilin  dye  the  poles  stain 
intensely,  but  a  narrow  space  between  them  remains 
almost  uncolored.  This  peculiarity  is  very  marked,  and 
sharp  observation  is  required  to  observe  the  outline  of 
the  intermediate  substance.  The  bacillus  does  not  form 
spores,  and  does  not  stain  by  Gram's  method.  When 
examined  in  the  living  condition  it  is  found  to  be  motile. 

325 


326  PATHOGENIC  BACTERIA. 

The  cultures  upon  gelatin  plates  after  about  two  days 
appear  as  small  white  points.  The  deep  colonies  reach 
the  surface  slowly,  and  do  not  attain  any  considerable 
size.  The  gelatin  is  not  liquefied.  The  microscope 


FIG.   105. — Bacillus  of  chicken-cholera,  from  the  heart's  blood  of  a  pigeon ; 
x  1000  (Frankel  and  Pfeiffer). 

shows  the  colonies  to  be  irregularly  rounded  disks  with 
distinct  smooth  borders.  The  color  is  yellowish-brown, 
and  the  contents  are  granular.  Sometimes  there  is  a  dis- 
tinct concentric  arrangement. 

In  gelatin  puncture-cultures  a  delicate  white  line  occurs 
along  the  entire  path  of  the  wire.  When  viewed  through 
a  lens,  this  line  can  be  seen  to  consist  of  aggregated  mi- 
nute colonies.  If,  instead  of  a  puncture,  the  inocula- 
tion be  made  upon  the  surface  of  obliquely  solidified 
gelatin,  a  much  more  pronounced  growth  takes  place, 
and  along  the  line  of  inoculation  a  dry,  granular  coat- 
ing is  formed.  This  growth  is  quite  similar  to  that 
upon  agar-agar  and  blood-serum,  which  growths  are 


CHICKEN-CHOLERA.  327 

white,  shining,  rather  luxuriant,  and  devoid  of  charac- 
teristics. 

Upon  potato  no  growth  occurs  except  at  the  incubation- 
temperature.  It  is  a  very  insignificant,  yellowish-gray, 
translucent  film. 

The  introduction  of  pure  cultures  of  this  bacillus  into 
the  tissues  of  chickens,  geese,  pigeons,  sparrows,  mice, 
and  rabbits  is  sufficient  to  produce  the  disease.  Feed- 
ing chickens,  pigeons,  and  rabbits  with  material  in- 
fected with  the  bacillus  is  also  sufficient  to  produce  the 
disease  with  pronounced  intestinal  lesions.  Guinea-pigs 
usually  seem  immune,  though  they  succumb  to  very 
large  doses,  especially  when  given  intraperitoneally. 

The  autopsy  shows  that  when  the  bacilli  are  intro- 
duced subcutaneously  a  true  septicemia  results,  with  the 
addition  of  a  hemorrhagic  exudate  and  gelatinous  infil- 
tration at  the  seat  of  inoculation.  The  liver  and  spleen 
are  enlarged,  circumscribed,  hemorrhagic,  and  infiltrated 
areas  occur  in  the  lungs ;  the  intestine  shows  an  in- 
tense inflammation  with  red  and  swollen  mucosa,  and 
occasional  ulcers  following  small  hemorrhagic  spots. 
The  bacilli  are  found  in  all  the  organs.  If,  on  the  other 
hand,  the  disease  has  been  produced  by  feeding,  the 
bacilli  are  chiefly  to  be  found  in  the  intestine.  Pasteur 
found  that  when  pigeons  were  inoculated  into  the  pectoral 
muscles,  if  death  did  not  come  on  rapidly,  portions  of  the 
muscle  (sequestra)  underwent  degeneration  and  appeared 
anemic,  indurated,  and  of  a  yellowish  color. 

The  bacillus  of  chicken-cholera  is  one  whose  peculiar- 
ities can  be  made  use  of  for  protective  vaccination. 
Pasteur  discovered  that  when  cultures  are  allowed  to 
remain  undisturbed  for  several  months,  their  virulence 
is  greatly  lessened,  and  new  cultures  planted  from  these 
are  also  attenuated.  When  chickens  are  inoculated  with 
such  cultures,  no  other  change  occurs  than  a  local  in- 
flammatory reaction  by  which  the  birds  are  protected 
against  virulent  bacilli.  From  this  observation  Pasteur 
worked  out  a  system  of  protective  vaccination  in  which 


328  PATHOGENIC  BACTERIA. 

fowls  can  first  be  inoculated  with  very  weak,  then  with 
stronger,  and  finally  with  highly  virulent  cultures,  with 
a  resulting  protection  and  immunity.  Unfortunately, 
the  method  is  too  complicated  to  be  very  practical. 

The  bacillus  of  chicken-cholera  seems  not  only  to  be 
specific  for  that  disease,  but  seems  able,  when  properly 
introduced  into  various  other  animals,  to  produce  several 
different  diseases.  Indeed,  no  little  confusion  has  arisen 
in  bacteriology  by  the  description  of  what  is  now  pretty 
generally  accepted  to  be  this  very  bacillus  under  the 
various  names  of  bacillus  of  rabbit-septicemia  (Koch), 
Bacillus  cuniculicida  (Fliigge),  bacillus  of  swine-plague 
(Lofner  and  Schiitz),  bacillus  of  u  Wildseuche  "  (Hiippe), 
bacillus  of  "  Buffelseuche  "  (Oriste-Armanni),  etc. 

In  1885,  Salmon'  and  Smith  wrote  upon  a  bacillus 
which  caused  an  epidemic  disease  of  hogs  in  certain 
parts  of  the  United  States,  calling  it  the  bacillus  of 
swine-plague,  but  at  first  regarding  it  as  different  from 
the  disease  well  known  in  Europe.  This  bacillus  has, 
however,  now  come  to  be  regarded  as  identical  with 
that  of  chicken-cholera. 

The  bacillus  of  "hog-cholera"  of  Klein,  Salmon,  and 
Smith  seems  to  differ  from  the  one  described  in  a  few 
particulars.  It  is  actively  motile,  is  provided  with  numer- 
ous flagella,  and  produces  upon  potato  a  straw  color 
which  may  turn  dark  when  old.  It  is  said  to  resemble 
very  closely  the  Bacillus  coli  communis,  and  it  is  thought 
by  Smith  to  be  a  close  ally  of  the  Bacillus  typhi  murium 
of  Corner. 


CHAPTER   VIII. 
MOUSE-SEPTICEMIA. 

IN  1878,  during  his  investigations  upon  the  infectious 
traumatic  diseases,  Koch  observed  that  when  a  minute 
amount  of  putrid  blood  or  of  meat-infusion  was  injected 
into  mice  the  animals  died  of  a  septicemia  caused  by  the 
multiplication  in  their  blood  of  a  minute  bacillus  to 
which  he  gave  the  name  "Bacillus  der  Mausesepticamie  " 
(Fig.  106). 


FIG.  106. — Bacillus  of  mouse-septicemia,  from  the  blood  of  a  mouse;  x  1000 
(Frankel  and  Pfeiffer). 

In  1885  the  bacillus  was  again  brought  into  promi- 
nence by  Loffler  and  Schiitz,  who  found  a  very  similar, 

329 


330  PATHOGENIC  BACTERIA. 

perhaps  identical,  organism  in  the  erysipelatous  disease 
which  attacks  the  swine  of  many  parts  of  Europe. 

There  seem  to  be  certain  slight  morphological  and 
developmental  differences  between  these  two  organisms, 
but  Baumgarten,  Gunther,  Sternberg,  and  others  have 
regarded  them  as  insufficient  for  the  formation  of  sepa- 
rate species,  and  have  boldly  described  the  organisms  as 
identical.  The  described  differences  are,  indeed,  so  very 
small  that  I  think  it  well  to  follow  in  the  path  of  the 
observers  mentioned,  pointing  out  in  the  description 
such  points  of  difference  as  may  arise. 

The  bacilli  are  extremely  minute,  measuring  about 
i.o  x  0.2  p  (Sternberg).  Fliigge,  Frankel,  and  Eisenberg 
find  the  Bacillus  erysipelas  suis  somewhat  shorter  and 
stouter  than  that  of  mouse-septicemia :  there  seems  to 
be  a  division  of  opinion  upon  this  point. 

Sporulation  has  been  described  by  some  observers,  but 
nothing  definite  seems  to  be  known  upon  this  point. 

Motility  is  ascribed  by  some  (Schottelius  and  Frankel) 
to  the  Bacillus  erysipelas  suis,  and  is  denied  to  the  bacillus 
of  mouse-septicemia  by  others.  The  truth  seems  to  be 
that  the  motility  of  both  organisms  is  a  matter  of  doubt. 

No  flagella  have  been  demonstrated  upon  the  bacillus. 
It  grows  quite  well  both  at  the  room-temperature  and  at 
the  temperature  of  incubation.     It  can  grow  well  with 
or  without  oxygen,  but  perhaps  flourishes 
a  little  better  without  than  with  it. 

The  colonies  upon  gelatin  plates  can 
first  be  seen  on  the  second  or  third  day, 
then   appearing  as  transparent  grayish 
FIG.  107.— Colony     specks    with    irregular    borders,     from 
of  the  bacillus  of     which    many   branched    processes    ex- 
mouse-septicemia ;  x     tend  (Fig>  IO^    prankel  describes  them 
as    resembling   in   shape    the    familiar 
branched  cells  occupying  the  lacunae   of  bone.     When 
further  developed  the   colonies  flow  together   and   give 
the  plate  a  cloudy  gray  appearance.     The  gelatin  is  not 
liquefied. 


MOUSE-SEPTICEMIA.  33* 

In  gelatin  puncture-cultures  the  growth  is  quite  cha- 
racteristic, and  the  tendency  of  the  bacillus  to  grow 
anerobically  is  well  shown  (Fig.  108).  The  develop- 


FIG.   108. — Bacillus  of  mouse-septicemia:  gelatin  puncture-culture  three  and  a 
half  days  old  (Gunther). 

ment  takes  place  all  along  the  line  of  puncture,  but  is 
more  marked  below  than  at  the  surface.  The  growth 
takes  place  in  a  peculiar  form,  resembling  superimposed 
disks,  each  disk  separate  from  its  neighbors  and  consist- 
ing of  an  area  of  clouded  grayish  gelatin  reaching  almost 
to  the  walls  of  the  tube.  This  growth  develops  slowly, 
and  causes  a  softening  rather  than  an  actual  liquefaction 
of  the  gelatin. 

Upon  agar-agar  and  blood-serum  a  very  delicate,  trans- 
parent grayish  line  develops  along  the  path  of  the  needle. 

The  bacillus  grows  at  the  room-temperature,  but  much 
better  at  the  temperature  of  the  incubator. 

The  disease  affects  quite  a  variety  of  animals,  notably 
hogs,  rabbits,  mice,  pigeons,  and  sparrows.  The  guinea- 
pig,  which  is  generally  the  victim  of  laboratory  experi- 
ments, is  not  susceptible  to  it. 

When  mice  are  inoculated  with  a  pure  culture  of  this 
bacillus,  they  soon  become  ill,  lose  their  appetite,  mope 
in  a  corner,  and  are  not  readily  disturbed.  As  the  dis- 


332  PATHOGENIC  BACTERIA. 

ease  becomes  worse  they  assume  a  sitting  posture  with 
the  back  much  bent  ;  the  eyelids  are  glued  together  by 
adhesive  pus ;  and  when  death  comes  to  their  relief,  in 
the  course  of  forty  to  sixty  hours  after  inoculation,  they 
remain  sitting  in  the  same  characteristic  position. 

When  the  ears  of  rabbits  are  inoculated  with  the 
bacillus  from  cases  of  erysipelas  suis,  a  violent  inflam- 
matory edema  and  distinct  redness  occurs,  much  re- 
sembling erysipelas.  This  lesion  gradually  spreads,  in- 
volves the  head,  then  the  body  of  the  animal,  and  ulti- 
mately causes  death. 

When  swine  are  affected,  they  are  dull  and  weak,  and 
have  a  kind  of  paralytic  weakness  of  the  hind  quarters. 
The  temperature  is  elevated  ;  red  patches  appear  upon 
the  skin  and  swell  and  become  tender.  Death  follows  in 
two  or  three  days. 

In  all  animals  the  anatomical  changes  are  much  alike. 
The  disease  proves  to  be  a  septicemia,  and  the  bacilli  can 
be  found  in  all  the  organs,  especially  the  lungs  and  spleen. 
They  are  few  in  number  in  the  streaming  blood. 

As  the  organisms  stain  well  by  Gram's  method,  this 
stain  is  of  great  value  for  their  discovery  in  the  tissues, 
and  can  be  highly  recommended. 

Most  of  the  bacilli  occupy  the  capillary  blood-vessels  ; 
many  of  them  are  enclosed  in  leucocytes.  The  organs  in 
such  cases  do  not  appear  distinctly  abnormal,  except  the 
spleen,  which  is  considerably  enlarged.  The  mesenteric 
and  other  lymphatics  are  also  enlarged,  and  the  gastric 
and  intestinal  mucous  membranes  are  usually  inflamed 
and  mottled.  The  bacilli  also  occupy  the  intestinal  con- 
tents, and  Kitt,  who  discovered  them  in  this  position, 
points  out  that  the  infection  of  swine  probably  takes 
place  by  the  entrance,  along  with  the  food,  of  the  fecal 
matter  of  diseased  animals  into  the  alimentary  apparatus 
of  others. 

Pasteur,  Chamberland,  Roux,  and  others  have  worked 
upon  a  protective  vaccination  based  upon  the  attenuation 
of  the  virulence  of  the  organism  by  passing  it  through 


MOUSE-SEPTICEMIA.  333 

rabbits.  Two  vaccinations  are  said  to  be  necessary  to 
produce  immunity.  The  vaccinated  animals,  however, 
may  be  a  source  of  infection  to  others,  and  should  always 
be  isolated.  Klemperer  in  1892  found  that  the  blood- 
serum  of  immunized  rabbits  would  save  infected  mice 
into  which  it  was  injected. 

L,orenz  in  1894  found  an  antitoxic  substance  in  the 
blood  of  rabbits  immunized  to  the  disease.  The  effect 
of  its  injection  into  other  animals  is,  however,  only  a 
temporary  immunity. 


CHAPTER    IX. 
ANTHRAX. 

THE  disease  of  cattle  known  as  anthrax  or  "splenic 
fever"  is  of  infrequent  occurrence  in  this  country  and  in 
England.  In  France,  Germany,  Hungary,  Russia,  Persia, 
and  the  East  Indian  countries  it  is  a  dreaded  and  common 
malady  which  robs  herdsmen  of  many  of  their  valuable 
stock.  Siberia  perhaps  suffers  most,  the  disease  being  so 
exceedingly  common  and  malignant  as  to  deserve  the 
name  "Siberian  pest."  Certain  local  areas,  such  as  the 
Tyrol  and  Auvergne,  in  which  it  seems  to  be  constantly 
present,  serve  as  distributing  foci  from  which  the  disease 
spreads  rapidly  in  summer,  afflicting  many  animals,  and 
ceasing  its  depredations  only  with  the  advent  of  winter. 
It  seems  to  be  distinctly  a  disease  of  the  summer  season. 

The  animals  most  frequently  affected  are  cows  and 
sheep.  Among  our  laboratory  animals  white  mice, 
guinea-pigs,  and  rabbits  are  highly  susceptible ;  dogs, 
cats,  most  birds,  and  amphibians  are  almost  perfectly 
immune.  White  rats  are  infected  with  difficulty.  Man 
is  only  slightly  susceptible,  the  manifestation  of  the  dis- 
ease as  seen  in  the  human  species  being  different  from 
the  same  disease  in  the  lower  animals  in  that  it  is  usually 
a  local  affection — malignant  carbuncle — and  only  at  times 
gives  rise  to  a  general  infection. 

Anthrax  was  one  of  the  first  of  the  specific  diseases 
proven  to  be  caused  by  a  definite  micro-organism.  As 
early  as  1849,  Pollender  discovered  small  rod-shaped 
bodies  in  the  blood  of  animals  suffering  from  anthrax, 
but  the  exact  relation  which  they  bore  to  the  disease  was 
not  pointed  out  until  1863,  when  Davaine,  by  a  series  of 
interesting  experiments,  proved  to  most  unbiased  minds 

334 


ANTHRAX. 


335 


their  etiological  significance.  The  further  confirmation 
of  Davaine's  conclusions  and  actual  proof  of  the  matter 
rested  with  Pasteur  and  Koch,  who,  observing  that  the 
bacilli  bore  spores,  cultivated  them  successfully  outside 
the  body,  and  then  produced  the  disease  by  the  inocula- 
tion of  pure  cultures. 

The  anthrax  bacilli  (Fig.  109)  are  large  rods  with  a 


FIG.  109.- 


-Bacillus  anthracis  :  colony  three  days  old  upon  a  gelatin  plate ;  ad- 
hesive preparation;  x  1000  (Frankel  and  Pfeiffer). 


rectangular  form,  caused  by  the  very  slight  rounding  of 
the  corners.  They  measure  5-20  ft  in  length  and  are 
from  i  fJL  to  1.25  fJ.  in  breadth.  The  pronounced  tendency 
is  toward  the  formation  of  long  threads,  in  which,  how- 
ever, the  individuals  can  generally  be  made  out ;  at  times 
isolated  rods  occur.  In  the  threads  the  bacilli  seem  en- 
larged a  little  at  the  ends,  and  give  somewhat  the  appear- 
ance of  a  bamboo  cane.  The  formation  of  spores  is  pro- 
lific :  each  spore  has  a  distinct  oval  shape,  is  transparent, 
and  does  not  alter  the  contour  of  the  bacillus  in  which  it 
occurs.  Spores  are  generally  formed  in  the  presence  of 
oxygen  upon  the  surfaces  of  the  culture-media.  When  a 
spore  is  placed  under  favorable  conditions  for  its  devel- 
opment and  is  carefully  watched,  it  may  be  observed  to 


33^  PATHOGENIC  BACTERIA. 

increase  in  length  a  trifle,  then  to  undergo  a  rupture  at 
one  end,  from  which  the  new  bacillus  projects.  The 
spores  of  anthrax  (Fig.  no),  being  large  and  easily  ob- 


r , 


FIG.  no. — Bacillus   anthracis,  stained  to  show  the  spores;  x   1000   (Frankel 

and  Pfeiffer). 

tainable,  are  excellent  subjects  for  the  study  of  sporula- 
tion,  for  the  action  of  germicides  and  antiseptics,  and  for 
demonstration  by  stains.  When  dried  upon  threads  of 
silk  they  will  retain  their  vitality  for  several  years,  and 
are  highly  resistant  to  heat  and  disinfectants. 

Spores  of  anthrax  are  killed  by  five  minutes'  exposure 
to  a  temperature  of  100°  C.,  and  are  killed  in  five  minutes 
in  a  5  per  cent,  solution  of  carbolic  acid,  or,  at  least,  are 
deprived  of  their  vegetative  property  in  relation  to  cul- 
ture-media. It  is  said  by  some  that  spores  subjected  to 
5  per  cent,  carbolic  acid  can  germinate  when  introduced 
into  susceptible  animals.  Spores  are  also  killed  by 
simple  wetting  with  i  :  100,000  bichlorid-of-mercury  so- 
lution. 

The  bacilli  are  not  motile  and  are  not  provided  with 
flagella.  They  stain  well  with  ordinary  solutions  of  the 
anilin  dyes,  and  can  be  beautifully  demonstrated  in  the 
tissues  by  Gram's  method  and  by  Weigert's  fibrin  method. 


ANTHRAX.  337 

Picro-carmin,  followed  by  Gram's  method,  gives  a  beau- 
tiful and  clear  picture.  The  spores  can  be  stained  with 
carbol-fuchsin,  the  bacilli  decolorized  with  a  very  weak 
acid,  and  then  counter-stained  with  a  watery  solution  of 
methyl  blue. 

Upon  the  surface  of  gelatin  plate-cultures  the  bacillus 
forms  beautiful  and  highly  characteristic  colonies  (Fig. 
in).  To  the  naked  eye  they  appear  first  as  minute 


FIG.  in. — Bacillus  anthracis:  colony  upon  a  gelatin  plate  ;  x  100  (Frankel  and 

Pfeiffer). 

round  whitish  dots  occurring  upon  the  surface,  and  caus- 
ing liquefaction  of  the  gelatin  as  they  increase  in  size. 
Under  the  microscope  they  can  be  seen  in  the  gelatin  as 
egg-shaped,  slightly  brownish  granular  bodies,  not  attain- 
ing their  full  development  except  upon  the  surface,  where 
they  spread  out  into  flat,  irregular,  transparent  growths 
bearing  a  partial  resemblance  to  tufts  of  curled  wool. 
From  a  tangled  centre  large  numbers  of  curls  extend, 
each  made  up  of  parallel  threads  of  bacilli.  As  soon  as 
the  colony  attains  any  considerable  size  liquefaction  be- 
gins. These  colonies  make  beautiful  adhesive  prepara- 
tions. If  a  perfectly  clean  cover-glass  be  passed  once 
'through  a  flame  and  laid  carefully  upon  the  gelatin,  the 
22 


338  PATHOGENIC  BACTERIA. 

colonies  can  generally  be  picked  up  entire  when  the  glass 
is  removed.  Such  a  specimen  can  be  dried,  fixed,  and 
stained  in  the  same  manner  as  an  ordinary  cover-glass 
preparation. 

In  gelatin  puncture-cultures  the  growth  is  even  more 
characteristic  than  are  the  colonies.  The  bacilli  begin 
to  grow  along  the  entire  track  of  the  wire,  most  luxuri- 
antly at  the  surface,  where  oxygen  is  plentiful.  As  the 
growth  progresses  fine  filaments  like  bristles,  extend 
from  the  puncture  into  the  neighboring  gelatin  giving 
the  growth  somewhat  the  appearance  of  an  evergreen 
tree  inverted  (Fig.  112). 


FIG.  112. — Bacillus  anthracis  :  gelatin  puncture-culture  seven  days  old 
(Giinther). 

The  more  superficial  of  these  threads  reach  about  half- 
way to  the  sides  of  the  tube,  while  the  deeper  ones  are 
shorter  and  shorter,  until  near  the  apex  branches  cease. 
When  the  projections  are  pretty  well  developed  a  distinct 
surface-growth  will  be  discerned,  and  if  the  tube  be  tilted, 
one  can  observe  that  the  gelatin  beneath  it  has  liquefied. 
As  the  growth  becomes  older  the  liquefaction  increases, 
until  ultimately  the  entire  gelatin  is  fluid  and  the  growth 
is  precipitated. 

Upon  agar-agar  the  characteristics  are  few.  The 
growth  takes  place  all  along  the  line  of  inoculation  as 


ANTHRAX.  339 

a  slightly  translucent,  slightly  wrinkled  layer  with  irreg- 
ular edges,  from  which  sufficient  bacillary  threads  pro- 
ject to  give  it  a  ciliated  appearance  to  the  naked  eye. 
When  the  culture  is  old  the  agar-agar  turns  a  distinct 
brown.  Spore-formation  is  luxuriant  upon  agar-agar. 

On  potato  the  growth  is  white,  creamy,  sometimes 
rather  dry  in  appearance.  Sporulation  is  marked. 

Blood-serum  cultures  lack  peculiarities ;  the  culture- 
medium  is  slowly  liquefied. 

The  bacillus  only  grows  between  the  extremes  of  20° 
and  45°  C.,  best  at  37°  C.  The  exposure  of  the  organ- 
ism to  the  temperature  of  42-43°  C.  for  twenty-four  hours 
is  sufficient  to  destroy  its  virulence. 

The  culture-media  should  always  be  faintly  alkaline,  as 
anthrax  bacilli  will  not  grow  in  the  presence  of  any  free 
acid. 

The  micro-organism  under  consideration  is  a  parasitic 
microbe,  yet  is  one  which,  because  of  its  spores,  can,  in 
a  latent  form,  exist  without  the  animal  organism  until 
appropriate  conditions  for  its  natural  development  are 
presented. 

Ordinarily,  the  infection  takes  place  either  through  the 
respiratory  tract  or  through  the  alimentary  canal. 

Buchner  has  shown  that  when  animals  are  allowed 
to  inhale  anthrax  spores  they  die  of  typical  anthrax. 
The  spores  establish  themselves  in  the  alveoli  of  the 
lung,  penetrate  the  epithelium,  enter  the  vascular  sys- 
tem, and  soon  give  rise  to  typical  lesions.  Strange  to 
say,  the  appearance  caused  by  the  inhalation  of  the 
bacilli  in  their  perfect  form  is  entirely  different,  for  a 
rapid  multiplication  occurs  without  Sporulation,  and 
causes  a  violent  irritative  pneumonia  with  serous  or  sero- 
fibrinous  exudate  in  which  large  numbers  of  the  bacilli 
occur.  In  these  cases  there  may  be  no  general  infection. 

When  the  bacilli  are  taken  into  the  stomach  in  food 
they  meet  with  a  rapid  death  because  of  the  acidity  of 
the  gastric  juice.  Should  spores,  however,  be  ingested, 
they  are  able  to  endure  the  gastric  juice,  to  pass  into  the 


340  PATHOGENIC  BACTERIA. 

intestine,  and,  as  soon  as  proper  conditions  of  alkalinity 
are  encountered,  to  develop  into  bacilli.  They  develop 
rather  rapidly,  surround  the  villi  with  thick  networks 
of  bacillary  threads,  separate  the  epithelial  cells,  enter 
the  lymphatics,  and  thus  find  the  appropriate  environ- 
ment for  the  production  of  a  general  infection. 

Sometimes  the  bacillus  enters  the  body  through  a 
wound,  cut,  scratch,  or  fly-bite.  This  is  especially  the 
case  with  men  who  come  in  contact  with  diseased  cattle. 
As  has  already  been  pointed  out,  a  malignant  pustule 
is  apt  to  follow,  and  may  cause  death.  Men  whose 
occupations  bring  them  in  contact  with  skins  and  hair 
from  animals  dead  of  anthrax  are  not  only  liable  to 
wound-infection,  but  are  sometimes  the  subjects  of  a  pul- 
monary form  of  the  disease — "wool-sorter's  disease" — 
caused  by  the  inspiration  of  the  spores  attached  to  the 
wool. 

The  disease  as  we  see  it  in  the  laboratory  is  accom- 
panied by  few  but  marked  lesions.  The  ordinary  method 
of  inoculation  is  -to  cut  away  a  little  of  the  hair  from 
the  abdomen  of  a  guinea-pig  or  rabbit  or  the  root  of 
a  mouse's  tail,  make  a  little  subcutaneous  pocket  with 
a  snip  of  a  pair  of  sterile  scissors,  and  introduce  the 
spores  or  bacilli  from  a  pure  culture  upon  a  rather  heavy 
platinum  wire,  the  end  of  which  is  flattened,  pointed, 
and  perforated.  An  animal  inoculated  in  this  way  gen- 
erally dies,  according  to  the  species,  in  from  twenty-four 
hours  to  three  days.  The  symptoms  are  weakness,  fever, 
loss  of  appetite,  and  sometimes  a  bloody  discharge  from 
nose  and  bowels.  There  is  much  subcutaneous  edema. 
At  the  autopsy  very  little  change  is  observed  at  the  seat 
of  inoculation.  The  subcutaneous  tissue  beneath  it  for 
a  considerable  distance  around  is  occupied  by  a  peculiar 
colorless  gelatinous  edema  which  contains  the  bacilli. 
The  abdominal  cavity  shows  injection  and  congestion 
of  its  viscera.  The  spleen  is  considerably  enlarged,  is 
dark  in  color,  and  .of  mushy  consistence.  The  liver  is 
somewhat  enlarged.  When  the  thorax  is  opened,  the 


ANTHRAX.          9  341 

lungs  may  be  slightly  congested,  but  otherwise  no 
changes  are  to  be  found. 

When  the  various  organs,  which  present  no  appreciable 
changes  to  the  naked  eye,  are  subjected  to  a  microscopic 
examination,  the  appropriate  staining  methods  bring  out 
a  most  remarkable  and  beautiful  change.  The  capil- 
lary system  is  almost  universally  occupied  by  bacilli, 
which  extend  throughout  its  meshworks  in  long  threads. 
Most  beautiful  bundles  of  these  bacillary  threads  can,  at 
times,  be  found  in  the  glomeruli  of  the  kidney  and  in 
the  minute  capillaries  of  the  intestinal  villi.  In  the 
larger  vessels,  where  the  blood-stream  is  rapid,  the  bac- 
teria are  relatively  few,  so  that  the  burden  of  bacillary 
obstruction  is  borne  by  the  minute  vessels.  The  con- 
dition is  thus  seen  to  be  one  of  pure  septicemia,  and 
bacilli  can  be  secured  in  pure  cultures  from  the  blood 
and  tissues. 

The  susceptibility  of  the  anthrax  bacillus  to  the  influ- 
ence of  heat,  cold,  antiseptics,  etc.  not  only  permitted 
Buchner,  Behring,  and  others  to  produce  biological  curi- 
osities in  the  form  of  bacilli  unable  to  bear  spores  and 
robbed  of  their  pathogenic  powers,  but  also  suggested 
to  Pasteur  the  important  practical  measure  of  protective 
vaccination.  Pasteur  found  that  the  inoculation  of  non- 
virulent  bacilli  into  cows  and  sheep,  and  their  reinocula- 
tion  with  slightly  virulent  bacilli,  gave  them  the  ability 
to  withstand  the  action  of  highly  virulent  organisms. 
Iv6ffler,  Koch,  and  Gaffky,  however,  found  that  these 
immunized  animals  were  not  absolutely  protected  from 
intestinal  anthrax. 

The  methods  of  diminishing  the  virulence  of  the 
anthrax  bacilli  are  numerous.  Toussaint,  who  was  cer- 
tainly the  first  to  produce  immunity  in  animals  by  inject- 
ing them  with  sterile  cultures  of  the  bacillus,  found  that 
the  addition  of  i  per  cent,  of  carbolic  acid  to  blood  of 
animals  dead  of  anthrax  destroyed  the  virulence  of  the 
bacilli ;  Chamberland  and  Roux  found  it  removed  when 
o.  1-0.2  per  cent,  of  bichromate  of  potassium  was  added  to 


342  PATHOGENIC  BACTERIA. 

the  culture-medium  ;  Chauveau  used  atmospheric  pressure 
to  the  extent  of  six  to  eight  atmospheres  and  found  the 
virulence  diminished ;  Arloing  found  that  direct  sunlight 
operated  similarly ;  Lubarsch  found  that  the  inoculation 
of  the  bacilli  into  immune  animals,  such  as  the  frog,  and 
their  subsequent  recovery  from  its  blood,  diminishes  the 
virulence  markedly. 

Protection,  can  be  afforded  in  still  other  ways.  The 
simultaneous  inoculation  of  bacteria  not  at  all  related  to 
anthrax  will  sometimes  recover  the  animal,  as  Hiippe 
found.  Hankin  found  in  the  cultures  chemical  sub- 
stances, especially  an  albuminose,  which  exerted  a  pro- 
tective influence.  Chamberland  has  shown  that  pro- 
tective inoculation  by  Pasteur's  method  has  diminished 
the  death-rate  from  10  per  cent,  for  sheep  and  5  per 
cent,  for  cattle  to  about  0.94  per  cent,  for  sheep  and  0.34 
per  cent,  for  cattle,  so  that  the  utility  of  the  method  is 
scarcely  questionable.  In  1890,  Agata  and  Jasuhara 
showed  that  in  the  convalescents  from  anthrax  among 
their  experimental  animals  an  antitoxic  substance  was 
present  in  the  blood  in  such  quantities  that  i  :  800  parts 
per  body-weight  of  dog's  serum  containing  the  antitoxin 
would  protect  a  mouse.  Similar  results  have  been  at- 
tained by  Marchoux. 

Experiments  of  interest  have  been  performed  to  show 
that  the  natural  immunity  enjoyed  by  many  animals  can 
be  destroyed.  Behring  found  that  if  the  alkalinity  of  the 
blood  of  rats  was  diminished,  they  could  become  affected 
with  anthrax,  and  numerous  observers  have  shown  that 
when  anthrax  bacilli  and  unrelated  organisms,  such  as 
the  erysipelas  cocci,  Bacillus  prodigiosus,  and  Bacillus 
pyocyaneus,  are  simultaneously  introduced  into  immune 
animals,  the  immunity  is  destroyed  and  the  animals 
succumb  to  the  disease.  Frogs  have  been  made  to  suc- 
cumb to  the  disease  by  exposure  to  a  temperature  of  37° 
C.  after  inoculation.  Pasteur  destroyed  the  immunity  of 
fowls  by  a  cold  bath  after  inoculation. 

In  the  natural  order   of   events  anthrax  in  cattle  is 


ANTHRAX.  343 

probably  the  result  of  the  inhalation  or  ingestion  of  the 
spores  of  the  bacilli  from  the  pasture.  At  one  time 
much  discussion  arose  concerning  the  infection  of  the 
pasture.  It  was  argued  that,  the  bacilli  being  enclosed 
in  the  tissues  of  the  diseased  animals,  the  infection  of 
the  pasture  must  be  due  to  the  distribution  of  the  germs 
from  the  buried  cadaver  to  all  parts  of  the  field,  either 
through  the  activity  of  earth-worms,  whi^h  ate  of  the 
earth  surrounding  the  corpse  and  then  deposited  the 
spores  in  their  excrement  at  remote  areas  (Pasteur),  or  to 
currents  of  moisture  in  the  soil.  Koch  seems,  however, 
to  have  demonstrated  the  fallacy  of  the  theories  by  show- 
ing that  the  conditions  under  which  the  bacilli  find  them- 
selves in  buried  cadavers  are  exactly  opposed  to  those 
favorable  to  fructification  or  sporulation,  and  that  in  all 
probability  the  majority  of  bacteria  suffer  the  same  fate 
as  the  animal  cells,  and  disintegrate,  especially  if  the  ani- 
mal be  buried  at  a  depth  of  two  or  three  meters. 

Frankel  points  out  particularly  that  no  infection  of  the 
soil  by  the  dead  animal  could  be  worse  than  the  pollution 
of  its  surface  by  the  bloody  stools  and  urine,  rich  in 
bacilli,  discharged  upon  it  by  the  animal  before  death, 
and  that  in  all  probability  it  is  the  live,  and  not  the  dead, 
animals  that  are  to  be  blamed  as  sources  of  infection. 

As  every  animal  affected  with  anthrax  is  a  source  of 
danger  to  the  community  in  which  it  t lives,  to  the  men 
who  handle  it  as  well  as  the  animals  who  browse  beside 
it,  such  animals,  as  soon  as  the  diagnosis  is  made,  should 
be  killed,  and,  together  with  the  hair  and  skin,  be  burned. 
When  this  is  impracticable,  Frankel  recommends  that 
they  be  buried  to  a  depth  of  at  least  iJ^-2  meters,  so 
that  the  sporulation  of  the  bacilli  is  impossible.  The 
dejecta  should  also  be  carefully  disinfected  with  5  per 
cent,  carbolic-acid  solution. 

Of  course,  animals  can  be  infected  through  wounds. 
This  mode  of  infection  is,  however,  more  common 
among  men,  who  suffer  from  the  local  disease  mani- 
fested as  the  malignant  carbuncle,  than  among  animals. 


CHAPTER  X. 

TYPHUS  MURIUM. 

THE  Bacillus  typhi  murium  (Fig.  113),  which  created 
havoc  among  the  mice  in  his  laboratory,  causing  most 
of  them  to  die,  was  discovered  by  Iv6ffler  in  1889.  It 
is  a  short  organism,  somewhat  resembling  the  bacillus 
of  chicken-cholera.  It  is  rather  variable  in  its  dimen- 
sions, and  often  grows  into  long,  flexible  filaments.  No 


FIG.    113. — Bacillus   typhi   murium,  from   agar-agar;     x    1000   (Itzerott   and 

Niemann). 

sporulation  has  been  observed.  It  is  a  motile  organism, 
with  numerous  flagella,  like  those  of  the  typhoid-fever 
bacillus.  It  stains  well  with  the  ordinary  dyes,  but 
rather  better  with  Loffler's  alkaline  methylene  blue. 

Upon  gelatin  plates  the  deep  colonies  are  at  first  round, 
slightly  granular,  transparent,  and  grayish.  Later  they 
become  yellowish-brown  and  granular.  Superficial  col- 
onies are  similar  to  those  of  the  typhoid  bacillus.  In 

344 


TYPHUS  MURIUM.  345 

gelatin  punctures  there  is  no  liquefaction.  The  growth 
takes  place  upon  the  surface  principally,  where  a  grayish- 
white  mass  slowly  forms. 

Upon  agar-agar  a  grayish-white  development  devoid 
of  peculiarities  occurs. 

Upon  potato  a  rather  thin  whitish  growth  may  be 
observed  after  a  few  days. 

The  bacillus  grows  well  in  milk,  with  the  production 
of  an  acid  reaction,  but  without  coagulation. 

The  organism  is  pathogenic  for  mice  of  all  kinds, 
which  succumb  in  from  one  to  two  days  when  inoculated 
subcutaneously,  and  in  eight  to  ten  or  twelve  days  when 
'fed  upon  material  containing  the  bacillus.  The  bacilli 
multiply  rapidly  in  the  blood-  and  lymph-channels,  and 
cause  death  from  a  general  septicemia. 

Loffler  expressed  the  opinion  that  this  bacillus  might 
be  of  use  in  ridding  infested  premises  of  mice,  and  the 
results  of  its  use  for  this  purpose  have  been  highly  satis- 
factory. He  has  succeeded  in  ridding  a  field  so  infested 
as  to  be  useless  for  agricultural  purposes  by  saturating 
some  bread  with  bouillon  cultures  of  the  bacillus  and 
distributing  it  near  the  holes  inhabited  by  the  mice. 
The  bacilli  that  were  eaten  by  the  mice  not  only  killed 
them,  but  also  infected  others  which  ate  the  dead  bodies 
of  the  first  victims,  and  so  the  extermination  progressed 
until  scarcely  a  mouse  remained  in  the  field.  The  bacilli 
are  not  pathogenic  for  the  animals,  such  as  the  fox,  weasel, 
ferret,  etc.,  that  feed  upon  the  mice,  do  not  affect  man  in 
any  way,  and  so  seem  to  occupy  a  useful  place  in  agri- 
culture by  destroying  the  little  but  almost  invincible 
enemies  of  the  grain. 


INDEX. 


ABBE  condenser  and  oil-immersion 
lenses,  hints  as  to  the  use  of, 

73 

Acid,  carbolic,  value  of,  as  a  germi- 
cide, 100 
Acids  and  alkalies,  production  of, 

by  bacteria,  52 
Actinomyces  bovis,  208 
Actinomycosis,  208 
fungus  of,  209 

growth  of,  210 
resemblance  of,  to  tuberculosis, 

210 
Activity,  vital,  in  bacteria,  results 

of,  48 

Adhesion  preparation,  125 
Aerobic  bacteria,  45 
Agar-agar  as  a  culture-medium,  1 10 
advantages  of,  over  gelatin,  127 
preparation  of,  1 10 
sedimentation  of,  in 
Air,  bacteriologic   examination  of, 

138 

Hesse's  apparatus,  139 
Petri's  filter  for,  140 
Sedgwick's  expanded  tube, 

141 

value  of,  141 
micro-organisms  in,  138 
pathogenic  bacteria  in,  138 
Alexin,  69 
Alkaloids,  animal,  49 

putrefactive,  49 
Anaerobic  bacteria,  45 
cultivation  of,  130 
Anilin  dyes   and  bacteria,  affinity 

between,  77 
classification  of,  77 
employment  of,  in   study   of 
bacteria,  77 


dyes     for    bacteriological 
work,  78 
introduction    of,   in    1877,   by 

Weigert,  26 
Animals,     experimentation    upon, 

134 

inoculation  of,  with  bacteria,  135 
Anthrax,  334 

animals  most  frequently  affected 

by,  334 

antitoxin  of,  342 
bacillus  of,  335 
cultures  of,  337 
discovery  of,  334 
morphology  of,  335 
pathogeny  of,  339 
resistant  powers  of,  3?.! 
staining  of,  335 
susceptibility  of,  to  heat,  cold, 

etc.,  341 

foci  for  the  distribution  of,  334 
immunity  to,  experiments  in  de- 
struction of,  341 
in  cattle,  how  acquired,  343 
measures  to  prevent  the  spread 

of,  343 
means  by  which  infection  takes 

place,  339 
means     of    protecting     animals 

against,  341,  342 
microscopic  examination  of  the 

various  organs  in,  341 
spores,  335,  336,  339 

resistant  powers  of,  336 
symptomatic,  248 
bacillus  of,  249 
cultures  of,  250 
staining  of,  249 
precautions  to  be  observed  in, 
253 

347 


348 


INDEX. 


Anthrax,    symptomatic,   protective 
inoculations  in,  251 

statistics  of,  253 
Antiabrin,  70 
Antiricin,  70 
Antisepsis,  origin  of,  26 
Antiseptic   value   of  some   of  the 

principal  germicides,  98 
Anti-streptococcus  serum,  161 
Antitoxic  serum,  232 

of  tetanus,  241 
Antitoxin  of  anthrax,  342 
of  cholera,  279,  280 
of  diphtheria,  230 
of  tetanus,  241 
theory  of  immunity,  69 
Antitoxins,  70 

action  of,  upon  bacteria,  7 1 
specific  for  one  disease  only,  72 
Arnold's  steam  sterilizer,  93 
Aromatics,  production  of,  by  bac- 
teria, 53 
Arthrospores,  35 
Ascococcus,  37 
Asiatic  cholera,    spirillum  of,  268, 

269 

Atmosphere    as    a   factor    in    the 
causation    of    suppuration, 

150 

bacteria  in,  1 50 
germs  in,  number  of,  141 
Autoclave,  94 

BACILLI,  division  of,  38 

morphology  of,  38 

motility  of,  31 

Bacillus  anthracis,  335 

colony  of,  124 

gelatin  puncture-culture  of,  126 
spores  of,  336 
coli  communis,  164,  255,  258 

cultures  of,  260,  261 
colon.     See   Bacillus  coli  com- 
munis. 

comma,  discovery  of,  29 
diphtherias,  220,  224,  225 

cover-glass  preparations  of,  221 
growth  of,  222 
morphology  of,  221 


Bacillus  diphtherias,  staining  of,  221 

toxin  elaborated  by,  229 
influenzas,  309 
Klebs-Loffier,  220 
leprae,  194 
growth  of,  195 
staining  of,  194 
liquefaciens   parvus,   colony   of, 

123 
mallei,  199 

cultivation  of,  200,  201 
staining  of,  202 

in  sections  of  tissue,  203 
Kiihne's  method,  202 
Lb'ffler's  method,  202 
mesentericus    vulgatus,    gelatin 

puncture-culture  of,  126 
muscoides,  colony  of,  124 
mycoides,  gelatin   puncture-cul- 
ture of,  126 

oedema  maligni,  312,  314 
of  bubonic  plague,  318 
of  chicken-cholera,  325 
of  fowl-tuberculosis,  190 
growth  of,  191 
staining  of,  191 
of  Friedlander,  303 
of  hog-cholera,  328 
of    malignant     edema,     gelatin 

puncture-culture  of,  126 
of  mouse-septicemia,  329,  331 
of  pseudo-diphtheria,  228 
of  pseudo-tuberculosis,  192 
of  rhinoscleroma,  219 
of  symptomatic  anthrax,  249 
inoculation  with,  251 
virulence  of,  251 
of  syphilis,  206 
of  typhoid  fever,  254 
pneumonias,  303 
cultures  of,  304 
pathogeny  of,  305 
polypiformis,  colony  of,  123 
pyocyaneus,  162 
pyogenes  fcetidus,  164 
radiatus,  colony  of,  123 

gelatin  puncture-culture  of,  126 
tetani,  235 

colony  on  gelatin,  237 


INDEX. 


349 


Bacillus  tetani,  cultures  of,  238 
distribution  of,  in  nature,  238 
isolation  of,  236 
means  of  entrance  into  animal 

organism,  239 
puncture-culture  of,  236 
resistant  powers  of,  237 
tuberculosis,  blood-serum  culture 

of,  177 
channels   by  which   it  enters 

the  organism,  181 
chemotactic  property  of,  184 
difficulty  in  staining,  171 
infection  by,  180 

through  the  gastro-intestinal 

tract,  182 

through  the  placenta,  181 
through  the  respiratory  tract, 

182 

through    the    sexual    appa- 
ratus, 183 

through  wounds,  183 
isolation  of,  by  Koch,  170 
pure  cultures  of.  178 
staining  of,  Ehrlich's  method, 

172 

Koch's  method,  172 
toxic  products  of,  187,  188 
typhi,  255 

abdominalis,  258 

gelatin  puncture-culture  of, 

126 

and  bacillus  coli  communis, 
resemblance  between,  260, 
261 

cultures  of,  257-260 
distribution  of,  in  nature,  256 
means   of   entrance   into    the 

body,  261 

morphology  of,  254 
murium,  344 
cultures  of,  344 
pathogenesis  of,  345 
staining  of,  344 
resistant  powers  of,  257 
staining  of,  256 

in  sections,  256 

typhoid,  means  of  entrance  into 
the  body,  261 


Bacilius  typhosus,  165 
Bacteria,  absence  of,  from  normal 
body-juices  and  tissues,  43 
action  of  antitoxins  upon,  71 
aerobic,  45 
anaerobic,  45 

cultivation  of,  130 
Botkin's  method,  133 
Buchner's  method,  130 
Esmarch's  method,  130 
Frankel's  method,  131 
Gruber's  method,  131 
Hesse's  method,  130 
Liborius'  method,  130 
Ravenel's  method,  132. 
Roux's  method,  133 
facultative,  45 
optional,  45 
and  anilin  dyes,  affinity  between, 

77 

and  spores,  difference  between,  35 

biology  of,  43 

changes  in  cell-walls  of,  31 

changes  undergone  by,  in  process 
of  staining,  74 

chemical  analysis  of,  30 

chromogenesis  of,  50 

chromogenic,  50 

classification  of,  40 

Cohn's  morphological,  42 

colonies   of,    appearance    under 

the  microscope,  123 
in    tubes,    Esmarch's    instru- 
ment for  counting,  145 

cover-glass  preparations  for  ex- 
amination of,  78 

cultivation  of,  106 

development  of,  in  liquids,  125 

distribution  of,  43 

examination  of,  in  solid  or  semi- 
solid  cultures,  76 

growing,  apparatus  for  examina- 
tion of,  76 

growth  of,  conditions  influencing, 

45 

electricity,  47 
light,  46 
moisture,  46 
movement,  47 


350 


INDEX. 


Bacteria,  growth  of,  conditions  in- 
fluencing :  nutriment,  45 
oxygen,  45 
reaction,  46 
temperature,  47 
in  gelatin,  126 
in  air,  43,  1 50 

determination  of,  139 
number  of,  141 
quantitative    estimation    of, 

J39 

in  body-juices  and  tissues  a  sign 

of  disease,  43 
influence  of  anilin  dyes  on,  30 

of  nuclear  stains  on,  30 
in  ice,  145 
injections  of,  into  animals,  134, 

135 

in  soil,  44,  147 
estimation  of  the  number  of, 

148 

in  tissue,  Gram's  method  of  stain- 
ing, 82 
introduction  of,  into  animals,  by 

injection,  134,  135 
in  water,  44 

apparatus  for  counting,  143 
filtration  as  a  means  of  dimin- 
ishing the  number  of,  146 
quantitative  determination  of, 

143 

isolation  of,  117 

liquefaction  of  gelatin  by,  51 

locomotory  powers  of,  31 

measurement  of,  88 

methods  of  cultivating,  117 
Esmarch  tubes,  121 
Petri  dishes,  121 
plate-cultures,  118 
of  observing,  73 

microscopic  examination  of,  123 

morphology  of,  36 

multiplication  of,  33 

non-chromogenic,  50 

non-pathogenic,  53 

of  specific  diseases,  149 

organization  of,  40 

parasitic,  48 

pathogenic,  53 


Bacteria,    pathogenic,   in  the    air, 

138 
means    of   entrance    into   the 

tissues,  54 
photographing  of,  89 
production  of  acids  and  alkalies 

by,  52 

of  aromatics  by,  53 
of  disease  by,  53 
of  gases  by,  52 
of  odors  by,  52 
of  phosphorescence  by,  53 
rate  of  development  of,  33 
recognition  of,  137 
reduction  of  nitrites  by,  53 
results  of  vital  activity  in,  48 
size  of,  32 

stained  or  unstained,  examina- 
tion of,  74 
staining  of,  in  sections  of  tissue, 

81 
study  of,  in  the  stained  condition, 

77 
that  do    not    stain    by   Gram's 

method,  84 
unstained,  method  of  examining, 

75 

weight  of,  33 
Bacteriologic  examination   of  air, 

value  of,  141 
of  soil,  147 
of  water,  143 

Bacteriology,  history  of,  17 
Bacterium,  38 

definition  of,  30 
Beef-peptone     in    preparation    of 

bouillon,  107 
Beggiatoa,  39 
Benches,  glass,  for  use  in  making 

plate-cultures,  120 
Binary  division,  33 

results  of,  33 

Birds,  susceptibility  of,  to  experi- 
mental inoculation  with  tu- 
bercle bacilli,    191 
Black-leg.  See  Anthrax,  symptom- 
atic. 
Blood-serum  as  a  culture-medium, 

112 


INDEX. 


351 


Blood-serum,  Koch's  apparatus  for 
coagulating   and  sterilizing, 

H3 
mixture,  Lb'rHer's,  114 

Body-juices,  antibactericidal  action 
of,  69 

Botkin's  apparatus  for  making  an- 
aerobic plate-cultures,  133 

Bouillon  as  a  culture-medium,  106 
preparation  of,  106 

Brownian  movement,  32 

Bubonic  plague,  318 
bacillus  of,  319 
cultures  of,  319 
discovery  of,  29 
pathogen esis  of,  320 

Buchner's  method  of  making  an- 
aerobic cultures,  130 

CARBOL-FUCHSIN,  86 
Carbolic  acid,  value  of,  as  a  germi- 
cide, 100 

Carmin   and  hematoxylon,  efforts 
to  facilitate  observation  of 
bacteria  by  means  of,  77 
Cells,  phagocytic,  63 
Charbon  symptomatique.    See  An- 
thrax, symptomatic. 
Chemotaxis,  63 
Chicken-cholera,  325 
bacillus  of,  325 
cultures  of,  326 
pathogenesis  of,  327 
pneumococcus  of,  discovery  of, 

28 
Cholera,  266 

Asiatic,  spirillum  of,  267 
cultures  of,  270-273 
staining  of,  270 
effect  of  vaccination  in  prevention 

of,  279 

immunity  to,   attempts    to    pro- 
duce, 278 
reasons  for,  275 
infectious  nature  of,  267 
spirillum   of,   in   drinking-water, 

means  of  detecting,  278 
pure   cultures   of,  Schottelius' 
method  of  securing,  271 


Cholera  spirillum,  toxic  products  of 

the  metabolism  of,  274 
theories  as  to  the  cause  of,  276 
Cilia,  31 
Cladothrix,  39 
Closet,  hot-air,  92 
Clostridium,  34 
Clothing,  disinfection  of,  103 
Cocci,  36 

morphology  of,  36 
Cohn's  classification  of  the  bacteria, 

41,  42 

Comma  bacillus,  discovery  of,  29 
Cotton,  sterile,  value  of,  in  bacte- 
riological work,  92 
Cover-glass  forceps,  80 
preparations  for  general  examina- 
tion, 78 
Gram's   method   for  staining, 

84 
method  of  fixing  material  for 

examination,  79 
Cover-glasses,  cleaning  of,  79 
Cultivation  of  bacteria,  106 
Culture-media,  106 
agar-agar,  no 
blood-serum,  112 
bouillon,  106 
Dunham's  solution,  116 
gelatin,  109 
glycerin  agar-agar,  112 
liquid,  best  means  of  keeping, 

107 
development    of   bacteria   in, 

125 

litmus  milk,  1 1 5 
LofHer's    blood-serum    mixture, 

114 

milk,  115 

peptone  solution,  116 
potatoes,  114 
potato-juice,  115 
sterilization  of,  93 
Cultures,  anaerobic,  various  meth- 
ods for  making,  130-133 
plate-,  118 
puncture-,  125 

in  gelatin,  various  appearances 
of,  126 


352 


INDEX. 


Cultures,  "pure,"  117 

method  of  making,  124 
solid  or  semi-solid,  examination 

of  bacteria  in,  76 
stroke-,  125 
study  of,  117 
Culture-tubes,    method    of   filling, 

1 08 

method  of  inoculating,  119 
Czenzynke's  staining  fluid,  310 

DAVAINE'S    classification     of    the 

bacteria,  41 

Dejecta,  sterilization  of,  101 
Digestive  tract,   entrance  of  bac- 
teria into,  54 

Diphtheria,  antitoxic  serum  in  treat- 
ment of,  234 
antitoxin,  230 

preparation  of,  230 
bacillus  of,  220,  224,  225 
discovery  of,  29 
toxin  elaborated  by,  229 
bacteriologic  diagnosis  of,  223 
in  man  and  in  animals,  228 
pseudo-,  bacillus  of,  229 
susceptibility  of  different  animals 

to,  227 

Diplococci,  36,  37 
Diplococcus  pneumonias,  298 
cultures  of,  299 
morphology  of,  298 
pathogenesis  of,  301 
Disease,  production  of,  by  bacteria, 

53 

Disease-germs,  isolation  and  culti- 
vation of,  28 

Diseases,  acute  inflammatory,  bac- 
teria of,  149 
chronic    inflammatory,    bacteria 

of,  169 

specific,  bacteria  of,  149 
toxic,  220 
Dishes,  Petri,  121 
Disinfection  of  clothing,  103 
of  furniture,  103 
of  patients,  104 
of  the  air  of  the  sick-room,  101 
of  the  skin,  99 


Disposal  of  the  bodies  of  persons 
dead  of  infectious  diseases, 
105 

Division,  binary,  of  bacteria,  33 
results  of,  33 

Drinking-water,  means  of  detecting 
cholera  spirilla  in,  278 

Dunham's  solution  as  a  culture- 
medium,  116 

Dyes,  anilin,  classification  of,  77 
introduction    of,  in     1877,   by 

Weigert,  26 
use  of,  in  study  of  bacteria,  77 

EDEMA,  malignant,  312 
bacillus  of,  312 

cultures  of,  314 

pathogenesis  of,  313 

staining  of,  314 
Ehrlich's  method  of  demonstrating 

the    presence     of    tubercle 

bacilli  in  sputum,  173 
of  staining  tubercle  bacilli  in 

sections  of  tissue,  175 
solution,  82 
Electricity,  influence  of,  on  growth 

of  bacteria,  47 
Endocarditis,  ulcerative,  production 

of,  by  injection  of  staphylo- 

coccus  pyogenes  aureus,  1 56 
Endospores,  34 
Enzymes,  tryptic,  51 
Erysipelas,  streptococcus  of,  1 59 
Esmarch  tubes,  121 
Esmarch's  instrument  for  counting 

colonies  of  bacteria  in  tubes, 

H5 

method  of  making  anaerobic  cul- 
tures, 130 
Examination,  bacteriologic,  of  air, 

138 

of  soil,  147 

of  water,  143 

Excreta,  disinfection  of,  102 
Exhaustion  theory  of  immunity,  62 
Experimentation  upon  animals,  134 

FARCIN  DU  BCEUF,  216 

streptothrix  of,  216,  217 


INDEX. 


353 


Fermentation,  48 

Fetus,   infection    of,    through    the 
placenta,    by    the     bacillus 
tuberculosis,  181 
Fever,   relapsing.     See   Relapsing 

fever. 

splenic.     See  Splenic  fever. 
typhoid.     See  Typhoid  fever. 
Filter,  Kitasato's,  96 

Pasteur-Chamberland,  95 
Petri's,  for  air-examination,  140 
Reichel's,  96 
Filters,  porcelain,  sterilization    of, 

77 
Filtration  of  culture-media,  95 

various  substances  used  for,  96 
of  toxins,  apparatus  for,  97 
Fiocca's  method  of  staining  spores, 

86 

Fission,  33 
Flagella,  31 
staining  of,  86 

conditions  essential  to  success 

in,  88 

Forceps,  cover-glass,  80 
Fowl-tuberculosis,  bacillus  of,  190 
Frankel's  instrument  for  obtaining 
earth   from    various   depths 
for  bacteriologic  study,  147 
method  of  making  anaerobic  cul- 
tures, 131 
Friedlander's   method  of  staining 

bacteria  in  tissue,  83 
pneumonia  bacillus,  303 
cultures  of,  304 
pathogeny  of,  305 
Funnel  for  filling  tubes  with  cul- 
ture-media, 108 
Furniture,  disinfection  of,  103 

GABBETT'S  method  of  demonstrat- 
ing the  presence  of  tubercle 
bacilli  in  sputum.  175 
Gases,  production  of,  by  bacteria, 

52 

Gelatin  as  a  culture-medium,  109 
growth  of  bacteria  in,  126 
growths,  microtome  sections  of, 
127 
23 


Gelatin,  liquefaction  of,  by  bacte- 
ria, 51 
Generation,  spontaneous,  doctrine 

of,  1 8 

"  Germ  theory  "  of  disease,  23 
Germicides,  chemical  action  of,  100 

values  of  different,  98 
Glanders,  198 
bacillus  of,  199 
cause  of,  198 

injections  of  mallein  in,  204 
Glassware,  sterilization  of,  91 
Glycerin    agar-agar  as   a  culture- 
medium,  112 

Gonococci,  cultivation  of,  166 
Gonococcus,  165 

in  urethral  pus,  165 
Gonorrhea,  165 

communication   of,   to  animals, 

167 
Gram's  method  of  staining  bacteria 

in  tissue,  82 

of  staining  cover-glass  prepa- 
rations, 84 
solution  for  staining  bacteria  in 

tissue,  83 

Gruber's  method  of  making  an- 
aerobic cultures,  131 

HANDS,  disinfection  of,  99 
Hanging-drop  method  of  examin- 
ing living  micro-organisms, 

75. 

Heat,  moist,  in  sterilization  of  ap- 
paratus used  in  experimen- 
tation, 91 

use  of,  in  sterilization  of  instru- 
ments, etc.,  91 

Hesse's    apparatus    for  collecting 

bacteria  from  the  air,  139 
method    of    making    anaerobic 
cultures,  130 

Heyroth's  instrument  for  counting 
colonies  of  bacteria  in  Petri 
dishes,  144 

History  of  bacteriology,  17 

Hog-cholera,  bacillus  of,  328 

Hot-air  closet,  92 

Humoral  theory  of  immunity,  67 


354 


INDEX. 


Hydrant-water,  number  of  bacteria 

in,  145 

Hydrophobia,  243 
and  tetanus,  parallelism  existing 

between,  244 
cure  for,  246 
incubation  period  of,  243 
treatment  of,    Pasteur's  system, 

246 

Hygienic  precautions  recommended 
for  preventing  the  spread  of 
tuberculosis,  180,  181 

ICE,  bacteria  in,  145 
Immunity,  acquired,  60 
and  susceptibility,  58,  59 
apparent,  61 

means  of  destroying,  60,  61 
natural,  59 
produced   by    antitoxins,  length 

of,  72 

theories  of,  62-72 
antitoxin,  69 
exhaustion,  62 
humoral,  67 
phagocytosis,  62 
retention,  62 

Incubating  oven  for  use  in  cultiva- 
tion of  bacteria,  129 
Infection,  bacterial,  through  the  di- 
gestive tract,  54 
through  the  placenta,  56 
through  the  respiratory  tract, 

55 
through  the  skin  and  superficial 

mucous  membranes,  56 
through  wounds,  56 
Influenza,  309 
bacillus  of,  309 

cultures  of,  310,  311 
discovery  of,  29 
staining  of,  310 
"  Infusorial  life,"  21 
Injection  of  bacteria  into  animals, 

134,  135 
Injections  of  tuberculin,  results  of, 

190 

Instruments,  disinfection  of,  100 
sterilization  of,  91 


Intra-abdominal  and  intrapleural 
injections  for  introduction 
of  bacteria  into  animals,  135 

Intravenous  injections  for  the  intro- 
duction of  bacteria  into  ani- 
mals, 134 

KITASATO'S  filter,  96 

"  Klatsch  praparat,"  125 

Koch-Ehrlich  method  of  demon- 
strating the  presence  of  tu- 
bercle bacilli  in  sputum, 

173 

Koch's  apparatus  for  coagulating 
and  sterilizing  blood-serum, 

H3 
steam  apparatus  for  sterilization 

of  culture-media,  93 
Kiihne's     carbol-methylene    blue, 
202 

LENSES,  high-power,  use  of,  74 
low-power,  use  of,  74 
oil-immersion,  use  of,  74 
Leprosy,  193 
anesthetic,  197 
bacillus  of,  194 
cause  of,  193 

discovery  of,  28 
nodes  of,  196 
Leptothrix,  33.  39 
Leuconostoc,  38 
Levelling    apparatus    for    pouring 

plate-cultures,  118 
Liborius'  method  of  making  anaer- 
obic cultures,  130 
Life,    spontaneous    generation    of, 

doctrine  of,  18 
Ligatures,  disinfection  of,  99 
Light,  influence  of,  on  growth  of 

bacteria,  46 

selection  of,  in  study  of  bacteria, 
by  means  of  the  microscope, 

74 
Liquid  culture-media,  development 

of  bacteria  in,  125 
Liquids,  sterilization  of,  95 
Listerism,  149 
origin  of,  27 


INDEX. 


355 


Litmus  milk  as  a  culture-medium, 

H5 
Loffler's  alkaline  methylene  blue, 

82 
blood-serum  mixture,  222 

as  a  culture-medium,  114 
method  of  staining  flagella,  86 
Lugol's  solution,  dilute,  for  staining 

bacteria  in  tissue,  83 
Lymphocytes,  63 

MADURA-FOOT,  212 
cause  of,  213 
streptothrix  of,  214 
Mallein,  203 

injections  of,  in  glanders,  204 
Measles,  316 
bacillus  of,  316 
cultures  of,  317 
discovery  of,  29 
staining  of,  316 
Meat-infusion,  107 
Merismopedia,  36,  37 
Methods  of  observing  bacteria,  73 
Methyl  violet,  antiseptic  value  of, 
destructive  and  inhibitory,  99 
Micrococci,  36 

Micrococcus  tetragenus,  165,  322 
cultures  of,  323 
pathogenesis  of,  324 
staining  of,  322 

Micro-organisms,  living,  hanging- 
drop  method  of  examination, 

75 

methods  of  destroying,  90 
on  the  skin,  150 

Microscope,  essential  features  of,  73 
Microtome     sections     of      gelatin 

growths,  127 

Milk  as  a  culture-medium,  115 
as  a  medium  for  the  cultivation 
of  the   bacillus  diphtherias, 
226 
Moisture,  influence  of,  on  growth 

of  bacteria,  46 
Mouse-holder,  136 
Mouse-septicemia,  329 
bacillus  of,  329 
cultures  of,  330 


Mouse-septicemia,  bacillus  of,  pa- 
thogen y  of,  332 
staining  of,  332 
Movement,  Brownian,  32 

influence  of,  on  growth  of  bac- 
teria, 47 
Mycetoma,  212 
streptothrix  of,  214 

cultures  of,  213 
Mycoderma,  126 
Myconostoc,  39 
Myco-phylaxin,  69 
Mycoprotein,  30 

composition  of,  30 
Myco-sozin,  69 

NITRITES,  reduction  of,  by  bac- 
teria, 53 

Nutriment,  influence  of,  on  growth 
of  bacteria,  45 

ODORS,  production  of,  by  bacteria, 
52 

Ophidiomonas,  40 

Osteomyelitis,  production  of,  by  in- 
jection of  the  staphylococcus 
pyogenes  aureus,  156 

Oxygen,  influence  of,  on  growth 
of  bacteria,  45 

PASTEUR-CHAMBERLAND  filter,  95 

Pasteur's  treatment  of  hydrophobia, 
246 

Patients,  disinfection  of,  104 

Peptone  solution  as  a  culture- 
medium,  116 

Pest,  Siberian.     See  Anthrax. 

Petri's  dishes,  121 

filter  for  air-examination,  140 

Phagocytes,  63 

Phagocytosis  theory  of  immunity,  62 

Phosphorescence,  production  of,  by 
bacteria,  53 

Phylaxins,  69 

Pigment-production,  50,  51 

Placenta,  entrance  of  bacteria 
through,  56 

Plague,  bubonic.  See  Bubonic 
plague. 


356 


INDEX. 


Plate-cultures,  118 

anaerobic,  Botkin's  apparatus  for 

making,  133 
apparatus  required  for  making, 

118 

drawbacks  to,  120 
method  of  making,  119 
Pneumobacillus,  304 
Pneumococcus,  304 
of  Frankel  and  Weichselbaum, 

164  . 

Pneumonia,  297 
bacillus  of,  304 
catarrhal,  305 
lobar  or  croupous,  297 
diplococcus  of,  298 
cultures  of,  299 
morphology  of,  298 
pathogenesis  of,  301 
tubercular,  305 
Pneumonias,  complicating,  306 

mixed,  306 

Potato  as  a  culture-medium,  114 
Potato-juice  as  a  culture-medium, 

H5 

Preparations,  cover-glass,  78,  79 

staining  of,  Gram's  method,  84 
Pseudo-diphtheria,  bacillus  of,  229 
Pseudo-tuberculosis,  191 

bacillus  of,  192 
Ptomaines,  definition  of,  49 
Pump-water,  number  of  bacteria  in, 

H5 

Puncture-cultures,  125 
gelatin,  various  appearances  of, 

126 

Pus,  urethral,  gonococcus  in,  165 
Putrefaction,  49 

QUARTER-EVIL.  See  Anthrax, 
symptomatic. 

RABBITS,  method  of  making  intra- 
venous injections  into,  135 

Rabies,  243.     See  Hydrophobia. 

Rauschbrand.  See  Anthrax, 
symptomatic. 

Ravenel's  method  of  making  an- 
aerobic cultures,  132 


Ray-fungus,  209 

Reaction,  influence  of,  on  growth 

of  bacteria,  46 
Reichel's  filter,  96 
Relapsing  fever,  307 
spirillum  of,  307 
pathogenesis  of,  308 
staining  of,  307 

Respiratory  tract,  entrance  of  bac- 
teria into,  55 
Results  of  vital  activity  in  bacteria, 

48 

chromogenesis,  50 
fermentation,  48 
liquefaction  of  gelatin,  51 
production   of   acids   and 

alkalies,  52 
production    of  aromatics, 

53 

production  of  disease,  53 
production  of  gases,  52 
production  of  odors,  52 
production  of  phosphores- 
cence, 53 
putrefaction,  49 
reduction  of  nitrites,  53 
Retention  theory  of  immunity,  62 
Rhinoscleroma,  219 

bacillus  of,  219 
River-water,  number  of  bacteria  in, 

,  H5 

Roux's  method  of  cultivating  an- 
aerobic bacteria,  133 

SAPROPHYTES,  48 
Sarcina,  36,  37 

Schottelius'  method  of  securing 
pure  cultures  of  the  cholera 
spirillum,  271 

Sedgwick's  expanded  tube  for  air- 
examination,  141 
Septic  diseases,  the,  307 
Serum,  anti-streptococcus,  161 
antitoxic,  of  anthrax,  342 
of  cholera,  279,  280 
of  diphtheria,  232 
of  tetanus,  241 

Siberian  pest.     See  Anthrax. 
Sick-room,  disinfection  of,  roi 


INDEX. 


357 


Skin  and  mucous  membranes,  en- 
trance  of  bacteria  through, 

56 

disinfection  of,  99 
Soil,  bacteria  of,  important,  148 

bacteriologic  examination  of,  147 
Solution,  Dunham's,  116 

Ehrlich's    anilin-water    gentian- 
violet,   173 
for  staining  bacteria  in  tissue, 

82 
Solutions,  disinfecting,  uselessness 

of,  in  the  sick-room,  101 
staining,  79 
Sozins,  69 
Spirilla,  39 

morphology  of,  39 

resembling  the  cholera  spirillum, 

281 

Spirillum  aquatilis,  295 
Berolinensis,  290 
Bonhoffi,  293 
Danubicus,  292 
Denecke,  284 
Dunbar,  291 
Kinkier  and  Pryor,  281 
Gamaleia,  287 
MetchnikofT,  287 
Milleri,  295 
terrigenus,  296 
Weibeli,  294 

I.  of  Wernicke,  293 

II.  of  Wernicke,  293 
Spirillum  aquatilis,  295 

Berolinensis,  290 

Bonhoffi,  293 

cholera  Asiatica,  268-274 
characteristics  of,  274 
cultures  of,  270-273 
distribution  of,  274 
in  drinking-water,  means  of 

detecting,  278 
inoculation  forms  of,  269 
production  of  indol  by,  274 
resistant  powers  of,  277 
staining  of,  270 
toxic  products  of  the  metab- 
olism of,  274 

Danubicus,  292 


Spirillum  Denecke,  284 

cultures  of,  285 
Dunbar,  291 
Finkler  and  Pryor,  281 
cultures  of,  282 
staining  of,  284 
Gamaleia,  287 
cultures  of,  288 
pathogenesis  of,  289 
MetchnikofT,  287 
Milleri,  295 

of  Asiatic  cholera,  268-274 
terrigenus,  296 
Weibeli,  294 

I.  of  Wernicke,  293 

II.  of  Wernicke,  293 
Spirochaeta,  39 

febris  recurrentis,  307 
Spiromonas,  40 
Spirulina,  40 
Splenic  fever,  334 
Spores,  34 

and  bacteria,  difference  between, 

35 
destruction    of,    by   intermittent 

sterilization,  94 
in  the  atmosphere,  1 50 
presence  of,  in  atmospheric  dust, 

90 

resistant  power  of,  35 
staining  of,  35,  85 

Fiocca's  method,  86 
Sporulation,  33,  34 

diagram  illustrating,  34 
Sputum,  tubercle  bacilli  in,  171 

demonstration  of,  172 
Sputum-cup,  sanitary,  102 
Staining    bacteria    in   sections   of 

tissue,  8 1,  82 
cover-glass  preparations,  Gram's 

method  for,  84 
flagella,  method  of,  86 
fluid,  Czenzynke's,  310 
of  tubercle  bacilli  in  sections  of 

tissue,  175,  176 
solutions,  stock,  79 
spores,  85 

Fiocca's  method,  86 
Staphylococci,  37 


358 


INDEX. 


Staphylococcus  epidermidis  albus, 

150 

"  golden,"  152 
pyogenes  albus,  151-154 

distribution  of,  in  nature,  1 52 
growth  of,  153,  1 54 
staining  of,  153 
citreus,  157 
Steam,  sterilization  of  culture-media 

by,  93 

superheated,  for  quick  steriliza- 
tion of  culture-media,  94 
Sterilization  and  disinfection,  90 
fractional,  93 
intermittent,  94 
of  air  of  the  sick-room,  101 
of   blood-serum,    Koch's    appa- 
ratus for,   113 
of  culture-media,  93 
of  dejecta,  101 

of  instruments,  etc.  used  in  ex- 
perimentation, 91 
of  liquids,  95 
of  porcelain  filters,  97 
of  surgical   dressings,  ligatures, 

etc.,  99 

Sterilizer,  Arnold's,  93 
hot-air,  92 
Koch's,  93 

Stock-solutions  for  staining,  79 
Streptococci,  36,  37 
Streptococcus  erysipelatis,  1 59 

as   a    therapeutic  measure  in 

treatment  of  tumors,  160 
pyogenes,  157 
growth  of,  158 
staining  of,  1 57 
Strepto-diplococcus,  37 
Streptothrix,  39 
Madurae,  214 

cultures  of,  213,  214 
of  farcin  du  bceuf,  216,  217 
Stroke-cultures,  125 
Subcutaneous  injections  for  the  in- 
troduction  of  bacteria  into 
animals,  134 
Suppuration,  149 

air  as  a  factor  in  the  causation 
of,  150 


Suppuration,  causes  of,  150 
Surgery,  antiseptic,  149 
Sutures,  disinfection  of,  99 
Symptomatic  anthrax,  248 
Syphilis,  205 
bacillus  of,  206 

staining  of,  205,  206 

TEMPERATURE,    influence    of,    on 

growth  of  bacteria,  47 
Tetanin,  240 
Tetano-toxin,  240 
Tetanus,  235 

and  hydrophobia,  parallelism  ex- 
isting between,  344 
antitoxic   serum   of,  preparation 

of,  241 

therapeutic  value  of,  242 
bacillus  of,  235 
cultures  of,  238 
discovery  of,  29 
distribution  of,  in  nature,  238 
pathology  of,  240 
susceptibility  to,  of  different  ani- 
mals, 239 

toxin  of,  preparation  of,  241 
Tetragenococci,  36,  37,  322 
Toxin  elaborated  by  the  bacillus 

diphtherias,  229 

Toxins,  rapid    filtration   of,    appa- 
ratus for,  97 
Toxo-phylaxin,  69 
Toxo-sozin,  69 
Tubercle  bacilli,  170 

channels  by  which  they  enter 

the  organism,  181 
cultivation  of,  176-179 
discovery  of,  28 
growth  of,  178 

in  sections  of  tissue,  175,  i?6 
methods  of  demonstrating 
the  presence  of,  175,  176 
.  in  sputum,  demonstration  of, 

172 

Ehrlich's  method,  173 
Gabbett's  method,  175 
Koch-Ehrlich  method,  173 
Ziehl's  method,  175 
pure  cultures  of,  178 


INDEX. 


359 


Tubercle  baccilli,  toxic  products  of, 

185,  187,  188 
Tubercles,  185,  186,  187 
Tuberculin.  188 
action  of,  188 
preparation  of,  189 
results  of  the  injection  of,  190 
Tuberculosis,  169 

bacillus  of,  170-188.  See  Tubercle 

bacilli. 

discovery  of,  28 
fowl-,  199 
gallinarum,  190 

hygienic      precautions      recom- 
mended for  preventing  the 
spread  of,  180,  181 
latent,  187 

microscopic  lesions  of,  183 
Tuberculous      patients,      sanitary 

sputum-cup  for  use  of,  102 
Tube,  Sedgwick's,  for  air-examina- 
tion, 141 

Tubes,  Esmarch,  121 
Tumors,  treatment  of,  by  inocula- 
tion with  the   streptococcus 
erysipelatis,  160 
Tyndall  on  the  "  germ  theory  "  of 

disease,  25 
Typhoid  fever,  254 
bacillus  of,  254 

cultures  of,  257-260 
discovery  of,  28 
resistant  powers  of,  257 
staining  of,  256 


Typhoid  fever,  comparative  im- 
munity of  animals  to,  263 

inoculation  experiments  on 
animals,  263 

prophylaxis  in,  265 

UNNA'S  method  of  staining  tubercle 
bacilli  in  sections  of  tissue, 
176 

VIBRIO,  39 

Vital  activity  in  bacteria,  results  of, 
48 

WATER,   bacteria  in,   quantitative 

determination  of,  143 
bacteriologic     examination      of, 

H3 
Wolfhiigel's  apparatus  for  counting 

colonies    of   bacteria    upon 

plates,  143 
Wounds,  unprotected,  entrance  of 

bacteria  into,  56 

YEAST-PLANT  as  the  cause  of  fer- 
mentation, discovery  of,  27 

ZIEHL'S  method  of  demonstrating 
the  presence  of  tubercle 
bacilli  in  sputum,  175 

Zooglea,  126 

Zoph's  classification  of  the  bacteria, 


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